SINGLE-CHAIN AND MULTI-CHAIN CHIMERIC POLYPEPTIDES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to: U.S. Patent Application Serial No. 62/816,683, filed March 11, 2019; U.S. Patent Application Serial No. 62/725,038, filed August 30, 2018; U.S. Patent Application Serial No. 62/817,244, filed March 12, 2019; U.S. Patent Application Serial No. 62/881,039, filed July 31, 2019; U.S. Patent Application Serial No. 62/724,969, filed August 30, 2018; U.S. Patent Application Serial No. 62/817,230, filed March 12, 2019; U.S. Patent Application Serial No. 62/725,043, filed August 30, 2018; U.S. Patent Application Serial No. 62/725,010, filed August 30, 2018; U.S. Patent

Application Serial No. 62/749,007, filed October 22, 2018; U.S. Patent Application Serial No. 62/746,832, filed October 17, 2018; U.S. Patent Application Serial No. 62/749,506, filed October 23, 2018; U.S. Patent Application Serial No. 62/817,241, filed March 12, 2019; and U.S. Patent Application Serial No. 62/881,088, filed July 31, 2019, each of which is incorporated hereby reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to the field of biotechnology, and more specifically, to single-chain and multi-chain chimeric polypeptides having a linker domain positioned between two target-binding domains that are useful for a variety of applications. BACKGROUND

Adoptive immunotherapy or cellular therapy requires the culture of immune cells obtained from a subject in vivo (and optionally genetic manipulation of the immune cells to express a chimeric antigen receptor or a T-cell receptor) before administration back into the subject. A sufficient number of immune cells is necessary in order to provide a therapeutic effect in the subject. In many examples, immune cells obtained from a subject need to be cultured for three or more weeks before a therapeutically effective number of immune cells can be obtained. In addition, many methods of culturing immune cells obtained from a subject in vitro require a layer of feeder cells, which requires subsequent purification or isolation of the immune cells before administration back to the subject.

SUMMARY

The present invention is based on the discovery that single-chain and multi-chain chimeric polypeptides having a linker domain positioned between two target-binding domains are effective in stimulating an immune cell, inducing or increasing proliferation of an immune cell, inducing differentiation of an immune cell, or treating a subject in need thereof (e.g., a subject having cancer or an aging-related disease or condition).

The present invention is also based on the discovery that the multi-chain chimeric polypeptides described herein promote the metabolism of the immune cells by increasing their aerobic glycolysis (Warburg Effect), oxidative phosphorylation, and mitochondrial reserve respiratory capacity to support their differentiation to effector cells and to enhance their effector-cell function(s).

In some aspects, provided herein are methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for the activation and proliferation of the natural killer cell or the T cell, wherein the first target-binding domain and the second target-binding domain are each independently selected from the group consisting of: a soluble interleukin, soluble cytokine protein, or soluble cell surface protein, an antigen-binding domain, a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor, and ligands of co-stimulatory molecules. In some embodiments, the first target-binding domain and the linker domain directly abut each other. In some embodiments, the single chain chimeric polypeptide further includes a linker sequence between the first target binding domain and the linker domain. In some embodiments, the linker domain and the second target-binding domain directly abut each other. In some embodiments, the single- chain chimeric polypeptide further includes a linker sequence between the linker domain and the second target-binding domain. In some embodiments, the first target-binding domain and the second target-binding domain directly abut each other. In some embodiments, the single-chain chimeric polypeptide further includes a linker sequence between the first target-binding domain and the second target-binding domain. In some embodiments, the second target-binding domain and the linker domain directly abut each other. In some embodiments, the single-chain chimeric polypeptide further includes a linker sequence between the second target-binding domain and the linker domain. In some embodiments, the first target-binding domain and the second target-binding domain bind specifically to the same antigen. In some embodiments, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments, the first target-binding domain and the second target-binding domain comprise the same amino acid sequence. In some embodiments, the first target-binding domain and the second target-binding domain bind specifically to different antigens. In some embodiments, one or both of the first target-binding domain and the second target- binding domain is an antigen-binding domain. In some embodiments, the first target- binding domain and the second target-binding domain are each an antigen-binding domain. In some embodiments, antigen-binding domain comprises a scFv or a single domain antibody.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, one or both of the first target-binding domain and the second target-binding domain bind to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD52, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a ULl6-binding protein, HLA-DR,

DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF- RII), a ligand of TGF-PRIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL- 3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-l 2, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD28.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin or cytokine protein. In some embodiments, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL- 2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein. In some embodiments, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin or cytokine receptor. In some embodiments, the soluble interleukin or cytokine receptor is a soluble TGF-b receptor II (TGF-PRII), a soluble TGF-PRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28. In some embodiments, one or both of the first target-binding domain and the second target-binding domain is a ligand of a co-stimulatory molecule. In some embodiments, the ligand of a co-stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, the linker domain is a soluble tissue factor domain. In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain does not include one or more of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. In some embodiments, the soluble human tissue factor domain does not include any of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. In some embodiments, the soluble tissue factor domain is not capable of binding Factor Vila. In some embodiments, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments, the single-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, the IgGl antibody construct includes at least one antigen-binding domain that binds specifically to the soluble tissue factor domain. In some embodiments, the linker domain is selected from the group consisting of: a kappa chain and a lambda chain. In some embodiments, the IgGl antibody construct is a monoclonal IgGl antibody, where both of the antigen binding domains in the monoclonal IgGl antibody bind specifically to the linker domain. In some embodiments, the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the linker domain. In some embodiments, the single-chain chimeric polypeptide further includes one or more additional target-binding domains at its N- and/or C- terminus. In some embodiments, the single-chain chimeric polypeptide includes one or more additional target-binding domains at its N-terminus. In some embodiments, one or more additional target-binding domains directly abuts the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments, the single chain chimeric polypeptide further includes a linker sequence between one of the at least one additional target-binding domains and the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide includes one or more additional target-binding domains at its C- terminus. In some embodiments, one of the one or more additional target-binding domains directly abuts the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide further includes a linker sequence between one of the at least one additional target- binding domains and the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide includes one or more additional target binding domains at its N-terminus and the C- terminus. In some embodiments, one of the one or more additional target-binding domains at the N-terminus directly abuts the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide further includes a linker sequence between one of the one or more additional target-binding domains at the N-terminus and the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments, one of the one or more additional target-binding domains at the C-terminus directly abuts the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide further includes a linker sequence between one of the one or more additional target-binding domains at the C- terminus and the first target-binding domain, the second target-binding domain, or the linker domain.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same antigen. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same epitope. In some

embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each comprise the same amino acid sequence. In some embodiments, the first target-binding domain, the second target- binding domain, and the one or more additional target-binding domains bind specifically to different antigens. In some embodiments, one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen-binding domain. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen-binding domain. In some embodiments, the antigen-binding domain comprises a scFv or a single domain antibody.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin,

CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF-b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-l 5, a receptor for IL- 17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28. In some

embodiments, one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. In some embodiments, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL- 18, IL-21 , PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP 16-binding protein. In some embodiments, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin or cytokine receptor. In some embodiments, the soluble interleukin or cytokine receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble

DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28. In some embodiments, one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a ligand of a co-stimulatory molecule. In some embodiments, the ligand of a co-stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4- 1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, the single-chain chimeric polypeptide further comprises a signal sequence at its N-terminal end. In some embodiments, the single-chain chimeric polypeptide lacks a signal sequence at its N- terminal end. In some embodiments, the single-chain chimeric polypeptide further comprises a peptide tag positioned at the N-terminal end or the C-terminal end of the single-chain chimeric polypeptide. In some embodiments, the single-chain chimeric polypeptide further includes a signal sequence at its N-terminal end. In some

embodiments, the single-chain chimeric polypeptide lacks a signal sequence at its N- terminal end.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, the contacting step is performed for a period of about 2 hours to about 20 days. In some embodiments, the contacting step is performed for a period of about 1 day to about 15 days. In some embodiments, the liquid culture medium is a serum-free liquid culture medium. In some embodiments, the liquid culture medium is a chemically-defined liquid culture medium. In some embodiments, the liquid culture medium comprises the single-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0.5: 1 to about 2: 1. In some embodiments, the liquid culture medium comprises the single-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0.8: 1 to about 1.2: 1.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, the NK cell or T cell was previously obtained from a subject. In some embodiments, the method further includes obtaining the NK cell or T cell from the subject prior to the contacting step. In some embodiments, the NK cell or T cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor. In some

embodiments, the method further includes, after the contacting step, introducing into the NK cell or the T cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. In some embodiments, the method further includes, before the contacting step, introducing into the NK cell or the T cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. In some embodiments, the method further includes, after the contacting step, isolating the NK cell or the T cell. In some embodiments, after the contacting step, the NK cell or the T cell has an increased level of expression or secretion of one or more proteins selected from the group consisting of: TNF-a, IFN-T, granzyme A, granzyme B, perforin, 2B4, CD8, CDl la,

CD 16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, CD 158a, CDl58b, CDl58e, CD178, CD226, CD253, NKG2A, NKG2C, NKG2D, LIR- 1 , LILR-B 1 , KIR2DL 1 , KIR3DL 1 , KIR2DL2, KIR2DL3 , CXCR3 ,

NKp30, NKp44, NKp46, NKG2D, DNAM-l, NKG2A, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-a, and IFN-g as compared to the level of expression or secretion of the one or more proteins prior to the contacting step.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, the method further includes, after the contacting step, administering the NK cell or the T cell to a subject in need thereof. In some embodiments, the subject has been identified or diagnosed as having an age-related disease or condition. In some embodiments, the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some embodiments, the subject has been identified or diagnosed as having a cancer. In some embodiments, the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject has been diagnosed or identified as having an infectious disease. In some embodiments, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus,

coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

In some aspects, provided herein are activated NK cell or T cells produced by any of the methods described herein that employ single-chain chimeric polypeptides. In some aspects, provided herein are pharmaceutical compositions that include such activated NK cells or T cell. In some aspects, provided herein are kits that include such pharmaceutical compositions. In some aspects, provided herein are methods of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof that include administering to the subject a therapeutically effective amount of the activated NK cell or the activated T cell produced by any of the methods described herein that employ single-chain chimeric polypeptides or the pharmaceutical composition that include such NK cells or T cell. In some embodiments, the subject has been identified or diagnosed as having a cancer. In some embodiments, the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject has been identified or diagnosed as having an aging-related disease or condition. In some embodiments, the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis,

Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some embodiments, the methods of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof include administering to the subject a therapeutically effective amount of the activated NK cell or the activated T cell produced by any of the methods described herein that employ single-chain chimeric polypeptides or the pharmaceutical composition that includes such activated NK cells or T cells. In some embodiments, the subject has been identified or diagnosed as having a cancer. In some embodiments, the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject has been identified or diagnosed as having an aging-related disease or condition. In some embodiments, the subject has been identified or diagnosed as having an aging-related disease or condition. In some embodiments, the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease,

spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some embodiments, the subject has been diagnosed or identified as having an infectious disease. In some embodiments, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

In some aspects, provided herein are kits that include (i) a single-chain chimeric polypeptide including a first target-binding domain, a linker domain, and a second target binding domain, wherein the first target-binding domain and the second target-binding domain are each independently selected from the group consisting of: a soluble interleukin or cytokine protein, an antigen-binding domain, a soluble interleukin or cytokine receptor, and ligands of co-stimulatory molecules; and (ii) an IgGl antibody construct including at least one antigen-binding domain that binds specifically to the linker domain. In some embodiments, the first target-binding domain and the linker domain directly abut each other. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the linker domain. In some embodiments, the linker domain and the second target- binding domain directly abut each other. In some embodiments, the single-chain chimeric polypeptide further includes a linker sequence between the linker domain and the second target-binding domain. In some embodiments, the first target-binding domain and the second target-binding domain directly abut each other. In some embodiments, the single-chain chimeric polypeptide further includes a linker sequence between the first target-binding domain and the second target-binding domain. In some embodiments, the second target-binding domain and the linker domain directly abut each other. In some embodiments, the single-chain chimeric polypeptide further includes a linker sequence between the second target-binding domain and the linker domain. In some embodiments, the first target-binding domain and the second target-binding domain bind specifically to the same antigen. In some embodiments, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments, the first target-binding domain and the second target-binding domain comprise the same amino acid sequence. In some embodiments, the first target-binding domain and the second target-binding domain bind specifically to different antigens. In some

embodiments, one or both of the first target-binding domain and the second target- binding domain is an antigen-binding domain. In some embodiments, the first target- binding domain and the second target-binding domain are each an antigen-binding domain. In some embodiments, the antigen-binding domain comprises a scFv or a single domain antibody.

In some embodiments of kits that include a single-chain chimeric polypeptide and an IgGl antibody construct, one or both of the first target-binding domain and the second target-binding domain bind to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL- 10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF-b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL- 3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-l 2, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a EILP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD28. In some embodiments, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. In some embodiments, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL- 2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L,

MICA, MICB, and a ULP16-binding protein. In some embodiments, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor. In some

embodiments, the soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28. In some embodiments, one or both of the first target-binding domain and the second target binding domain is a ligand of a co-stimulatory molecule. In some embodiments, the ligand of a co-stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l.

In some embodiments of kits that include a single-chain chimeric polypeptide and an IgGl antibody construct, the linker domain is a soluble tissue factor domain. In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain includes a sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain includes a sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain includes a sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain does not include one or more of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

In some embodiments, the soluble human tissue factor domain does not include any of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. In some embodiments, the soluble tissue factor domain is not capable of binding Factor Vila. In some embodiments, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments, the single-chain chimeric polypeptide does not stimulate blood coagulation in a mammal. In some embodiments of kits that include a single-chain chimeric polypeptide and an IgGl antibody construct, the IgGl antibody construct includes at least one antigen binding domain that binds specifically to the soluble tissue factor domain. In some embodiments, the linker domain is selected from the group consisting of: a kappa chain and a lambda chain. In some embodiments, the IgGl antibody construct is a monoclonal IgGl antibody, where both antigen-binding domains in the monoclonal IgGl antibody bind specifically to the linker domain. In some embodiments, the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the linker domain.

In some embodiments of kits that include a single-chain chimeric polypeptide and an IgGl antibody construct, the single-chain chimeric polypeptide further includes one or more additional target-binding domains at its N- and/or C-terminus. In some

embodiments, the single-chain chimeric polypeptide includes one or more additional target-binding domains at its N-terminus. In some embodiments, the one or more additional target-binding domains directly abuts the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments, the single chain chimeric polypeptide further includes a linker sequence between one of the at least one additional target-binding domains and the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide includes one or more additional target-binding domains at its C- terminus. In some embodiments, one of the one or more additional target-binding domains directly abuts the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide further includes a linker sequence between one of the at least one additional target- binding domains and the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide includes one or more additional target binding domains at its N-terminus and the C- terminus. In some embodiments, one of the one or more additional target-binding domains at the N-terminus directly abuts the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide further includes a linker sequence between one of the one or more additional target-binding domains at the N-terminus and the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments, one of the one or more additional target-binding domains at the C-terminus directly abuts the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide further includes a linker sequence between one of the one or more additional target-binding domains at the C- terminus and the first target-binding domain, the second target-binding domain, or the linker domain.

In some embodiments of kits that include a single-chain chimeric polypeptide and an IgGl antibody construct, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments, two or more of the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains bind specifically to the same epitope. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same antigen. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same epitope. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each include the same amino acid sequence. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to different antigens. In some embodiments, one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen-binding domain. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen-binding domain. In some embodiments, the antigen-binding domain includes a scFv or a single domain antibody. In some embodiments of kits that include a single-chain chimeric polypeptide and an IgGl antibody construct, one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3,

CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF-b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL- 17, a receptor for IL-l 8, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28. In some

embodiments, one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. In some embodiments, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL- 18, IL-21 , PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP 16-binding protein. In some embodiments, one or more of the first target-binding domain, the second target binding domain, and the one or more additional target-binding domains is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor. In some embodiments, the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28. In some embodiments, one or both of the first target- binding domain and the second target-binding domain is a ligand of a co-stimulatory molecule. In some embodiments, the ligand of a co-stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l. In some embodiments, the single-chain chimeric polypeptide further includes a peptide tag positioned at the N-terminal end or the C- terminal end of the single-chain chimeric polypeptide. In some embodiments, the single- chain chimeric polypeptide further includes a signal sequence at its N-terminal end. In some embodiments, the single-chain chimeric polypeptide lacks a signal sequence at its N-terminal end.

In some aspects, provided herein are methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium including: (1) an effective amount of a multi-chain chimeric polypeptide that includes: (a) a first chimeric polypeptide including: (i) a first target-binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgGl antibody construct including at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for the activation and proliferation of the natural killer cell or the T cell. In some embodiments, the first target- binding domain and the linker domain directly abut each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence between the first target-binding domain and the linker domain in the first chimeric polypeptide. In some embodiments, the linker domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence between the linker domain and the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further includes a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide. In some embodiments, the first target-binding domain and the second target-binding domain bind specifically to the same antigen. In some embodiments, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments, the first target-binding domain and the second target-binding domain include the same amino acid sequence. In some embodiments, the first target-binding domain and the second target-binding domain bind specifically to different antigens. In some embodiments, one or both of the first target-binding domain and the second target- binding domain is an antigen-binding domain. In some embodiments, the first target- binding domain and the second target-binding domain are each antigen-binding domains. In some embodiments, the antigen-binding domain includes a scFv or a single domain antibody.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium that includes first and second chimeric polypeptides and an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to a linker domain in one chimeric polypeptide, one or both of the first target- binding domain and the second target-binding domain bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD 137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR,

HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a EIL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF- DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF-b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD28. In some embodiments, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin or cytokine protein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. In some embodiments, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL- 2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein. In some embodiments, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor. In some

embodiments, the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28. In some embodiments, one or both of the first target-binding domain and the second target-binding domain is a ligand of a co-stimulatory molecule. In some embodiments, the ligand of a co-stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium that includes first and second chimeric polypeptides and an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to a linker domain in one chimeric polypeptide, the first chimeric polypeptide further includes one or more additional target-binding domain(s), where at least one of the one or more target-binding domain(s) is positioned between the linker domain and the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further includes a linker sequence between the linker domain and the at least one of the one or more target antigen-binding domain(s), and/or a linker sequence between the at least one of the one or more target antigen-binding domain(s) and the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further includes one or more additional target-binding domains at the N- terminal and/or C-terminal end of the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains. In some embodiments, the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence between the at least one of the one or more additional target- binding domains and the first target-binding domain. In some embodiments, at least one of the one or more additional target-binding domains is disposed at the N- and/or C- terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains is positioned between the linker domain and the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, he at least one additional target-binding domain of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence disposed between the at least one additional target- binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains, directly abuts the linker domain and/or the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further includes a linker sequence disposed (i) between the linker domain and the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium that includes first and second chimeric polypeptides and an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to a linker domain in one chimeric polypeptide, the second chimeric polypeptide further includes one or more additional target-binding domains at the N- terminal end and/or the C-terminal end of the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further includes a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further includes a linker sequence between at least one of the one or more additional target-binding domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium that includes first and second chimeric polypeptides and an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to a linker domain in one chimeric polypeptide, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains include the same amino acid sequence. In some embodiments, the first target-binding domain, the second target- binding domain, and the one or more additional target-binding domains each bind specifically to the same antigen. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same epitope. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each comprise the same amino acid sequence. In some embodiments, first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to different antigens. In some embodiments, one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen-binding domain. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen-binding domain. In some embodiments, the antigen-binding domain includes a scFv.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium that includes first and second chimeric polypeptides and an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to a linker domain in one chimeric polypeptide, one or more of the first target-binding domain, the second target-binding domain, and the one or more target binding domains bind specifically to a target selected from the group consisting of:

CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD 137, CD40, CD47, CD70, 0X40, IGF- 1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF-b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL- 3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-l 2, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a EILP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD3, and a receptor for CD28. In some embodiments, one or more of the first target binding domain, the second target-binding domain, and the one or more additional target binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. In some embodiments, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL- 7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA,

MICB, and a ULP16-binding protein. In some embodiments, one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor. In some embodiments, the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28. In some embodiments, the first chimeric polypeptide further comprises a peptide tag at the N- terminal end or the C-terminal end of the first chimeric polypeptide. In some one or both of the first target-binding domain and the second target-binding domain is a ligand of a co-stimulatory molecule. In some embodiments, the ligand of a co-stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l. In some embodiments, the first chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium that includes first and second chimeric polypeptides and an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to a linker domain in one chimeric polypeptide, the linker domain is a soluble tissue factor domain. In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain includes a sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain includes a sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain includes a sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain does not include one or more of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. In some embodiments, the soluble human tissue factor domain does not include any of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. In some embodiments, the soluble tissue factor domain is not capable of binding to Factor Vila. In some embodiments, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments, the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium that includes first and second chimeric polypeptides and an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to a linker domain in one chimeric polypeptide, the IgGl antibody construct includes at least one antigen-binding domain that binds specifically to the soluble tissue factor domain. In some embodiments, the linker domain is selected from the group consisting of: a kappa chain and a lambda chain. In some embodiments, the IgGl antibody construct is a monoclonal IgGl antibody, where both antigen-binding domains in the monoclonal IgGl antibody bind specifically to the linker domain. In some embodiments, the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the linker domain.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium that includes first and second chimeric polypeptides and an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to a linker domain in one chimeric polypeptide, the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (ILl5Ra) and a soluble IL- 15. In some embodiments, the soluble IL15 has a D8N or D8A amino acid substitution. In some embodiments, the human ILl5Ra is a mature full-length ILl5Ra. In some embodiments, the pair of affinity domains is selected from the group consisting of: barnase and barnstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. In some embodiments, the first chimeric polypeptide and/or the second chimeric polypeptide further includes a signal sequence at its N-terminal end. In some embodiments, the first chimeric polypeptide and/or the second chimeric polypeptide lacks a signal sequence at its N-terminal end.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium that includes first and second chimeric polypeptides and an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to a linker domain in one chimeric polypeptide, the contacting step is performed for a period of about 2 hours to about 20 days. In some embodiments, the contacting step is performed for a period of about 1 day to about 15 days. In some embodiments, the liquid culture medium is a serum-free liquid culture medium. In some embodiments, the liquid culture medium is a chemically-defined liquid culture medium. In some embodiments, the liquid culture medium comprises the multi-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0.5: 1 to about 2: 1. In some embodiments, the liquid culture medium comprises the multi-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0.8: 1 to about 1.2: 1. In some embodiments, the NK cell or T cell was previously obtained from a subject. In some embodiments, the method further includes obtaining the NK cell or T cell from the subject prior to the contacting step. In some embodiments, the NK cell or T cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor. In some embodiments, the method further includes, after the contacting step, introducing into the NK cell or the T cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. In some embodiments, the method further includes, before the contacting step, introducing into the NK cell or the T cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. In some embodiments, the method further includes, after the contacting step, isolating the NK cell or the T cell. In some embodiments, after the contacting step, the NK cell or the T cell has an increased level of expression or secretion of one or more proteins selected from the group consisting of: TNF-a, IFN-T, granzyme A, granzyme B, perforin, 2B4, CD8, CDlla, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, CDl58a, CDl58b, CDl58e, CD178, CD226, CD253, NKG2A, NKG2C, NKG2D, LIR-l, LILR-B1, KIR2DL1, KIR3DL1, KIR2DL2, KIR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-l, NKG2A, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-a, and IFN-g as compared to the level of expression or secretion of the one or more proteins prior to the contacting step. In some embodiments, the method further includes, after the contacting step, administering the NK cell or the T cell to a subject in need thereof.

In some embodiments of methods of promoting the activation and proliferation of a natural killer cell or a T cell that include contacting a natural killer cell or a T cell in a liquid culture medium that includes first and second chimeric polypeptides and an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to a linker domain in one chimeric polypeptide, the subject has been identified or diagnosed as having an age-related disease or condition. In some embodiments, the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis,

Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some embodiments, the subject has been identified or diagnosed as having a cancer. In some embodiments, the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma,

neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject has been diagnosed or identified as having an infectious disease. In some embodiments, the infectious disease is infection with human

immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

In some aspects, provided herein are activated NK cell or T cells produced by any of the methods described herein that employ multi-chain chimeric polypeptides. In some aspects, provided herein are pharmaceutical compositions that include such activated NK cells or T cell. In some aspects, provided herein are kits that include such pharmaceutical compositions. In some aspects, provided herein are methods of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof that include administering to the subject a therapeutically effective amount of the activated NK cell or the activated T cell produced by any of the methods described herein that employ multi-chain chimeric polypeptides or the pharmaceutical composition that include such NK cells or T cell. In some embodiments, the subject has been identified or diagnosed as having a cancer. In some embodiments, the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject has been identified or diagnosed as having an aging-related disease or condition. In some embodiments, the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis,

Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some embodiments, the methods of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof include administering to the subject a therapeutically effective amount of the activated NK cell or the activated T cell produced by any of the methods described herein that employ multi-chain chimeric polypeptides or the pharmaceutical composition that includes such activated NK cells or T cells. In some embodiments, the subject has been identified or diagnosed as having a cancer. In some embodiments, the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject has been identified or diagnosed as having an aging-related disease or condition. In some embodiments, the subject has been identified or diagnosed as having an aging-related disease or condition. In some embodiments, the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease,

spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some embodiments, the subject has been diagnosed or identified as having an infectious disease. In some embodiments, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

In some aspects, provided herein are kits that include: 1) a multi-chain chimeric polypeptide that includes: (a) a first chimeric polypeptide including: (i) a first target- binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgGl antibody construct including at least one antigen-binding domain that binds specifically to the linker domain. In some embodiments, the first target-binding domain and the linker domain directly abut each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence between the first target-binding domain and the linker domain in the first chimeric polypeptide. In some embodiments, the linker domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence between the linker domain and the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further includes a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide. In some

embodiments, the first target-binding domain and the second target-binding domain bind specifically to the same antigen. In some embodiments, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments, the first target-binding domain and the second target-binding domain include the same amino acid sequence. In some embodiments, the first target-binding domain and the second target-binding domain bind specifically to different antigens. In some embodiments, one or both of the first target-binding domain and the second target- binding domain is an antigen-binding domain. In some embodiments, the first target- binding domain and the second target-binding domain are each antigen-binding domains. In some embodiments, the antigen-binding domain includes a scFv or a single domain antibody.

In some embodiments of kits that include a multi-chain chimeric polypeptide and an IgGl antibody construct, one or both of the first target-binding domain and the second target-binding domain bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD 137, CD40, CD47, CD70, 0X40, IGF- 1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF-b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL- 3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD28. In some embodiments, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. In some embodiments, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL- 2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-l 7, IL-l 8, IL-21, PDGF-DD, SCF, FLT3L,

MICA, MICB, and a ULP16-binding protein. In some embodiments, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor. In some

embodiments, the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28. In some embodiments, one or both of the first target-binding domain and the second target-binding domain is a ligand of a co-stimulatory molecule. In some embodiments, the ligand of a co-stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l.

In some embodiments of kits that include a multi -chain chimeric polypeptide and an IgGl antibody construct, the first chimeric polypeptide further includes one or more additional target-binding domain(s), where at least one of the one or more target-binding domain(s) is positioned between the linker domain and the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further includes a linker sequence between the linker domain and the at least one of the one or more target antigen-binding domain(s), and/or a linker sequence between the at least one of the one or more target antigen-binding domain(s) and the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further includes one or more additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains. In some embodiments, the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence between the at least one of the one or more additional target-binding domains and the first target- binding domain. In some embodiments, at least one of the one or more additional target- binding domains is disposed at the N- and/or C-terminus of the first chimeric

polypeptide, and at least one of the one or more additional target-binding domains is positioned between the linker domain and the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the at least one additional target- binding domain of the one or more additional target-binding domains disposed at the N- terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the at least one additional target-binding domain of the one or more additional target- binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence disposed between the at least one additional target-binding domain and the first target- binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the at least one of the one or more additional target- binding domains positioned between the linker domain and the first domain of the pair of affinity domains, directly abuts the linker domain and/or the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further includes a linker sequence disposed (i) between the linker domain and the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains.

In some embodiments of kits that include a multi -chain chimeric polypeptide and an IgGl antibody construct, the second chimeric polypeptide further includes one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further includes a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further includes a linker sequence between at least one of the one or more additional target-binding domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of kits that include a multi -chain chimeric polypeptide and an IgGl antibody construct, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments, two or more of the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains bind specifically to the same epitope. In some two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains include the same amino acid sequence. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same antigen. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same epitope. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each include the same amino acid sequence. In some embodiments, the first target-binding domain, the second target- binding domain, and the one or more additional target-binding domains bind specifically to different antigens. In some embodiments, one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen-binding domain. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen-binding domain. In some embodiments, the antigen-binding domain includes a scFv.

In some embodiments of kits that include a multi -chain chimeric polypeptide and an IgGl antibody construct, one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF-b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL- 17, a receptor for IL-l 8, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a EILP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD3, and a receptor for CD28. In some embodiments, one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. In some embodiments, the soluble interleukin, soluble cytokine protein, or soluble cell surface receptor is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL- 12, IL-l 5, IL-l 7, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULPl6- binding protein. In some embodiments, one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor. In some embodiments, the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28. In some embodiments, one or both of the first target-binding domain and the second target binding domain is a ligand of a co-stimulatory molecule. In some embodiments, the ligand of a co-stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l.

In some embodiments of kits that include a multi -chain chimeric polypeptide and an IgGl antibody construct, the linker domain is a soluble tissue factor domain. In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain.

In some embodiments, the soluble human tissue factor domain includes a sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain includes a sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain includes a sequence that is at least 95s% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain does not include one or more of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

In some embodiments, the soluble human tissue factor domain does not include any of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. In some embodiments, the soluble tissue factor domain is not capable of binding to Factor Vila. In some embodiments, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments, the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

In some embodiments of kits that include a multi -chain chimeric polypeptide and an IgGl antibody construct, the IgGl antibody construct includes at least one antigen binding domain that binds specifically to the soluble tissue factor domain. In some embodiments, the linker domain is selected from the group consisting of: a kappa chain and a lambda chain. In some embodiments, the IgGl antibody construct is a monoclonal IgGl antibody, where both antigen-binding domains in the monoclonal IgGl antibody bind specifically to the linker domain. In some embodiments, the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the linker domain.

In some embodiments of kits that include a multi-chain chimeric polypeptide and an IgGl antibody construct, the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (ILl5Ra) and a soluble IL-15. In some embodiments, the soluble IL15 has a D8N or D8A amino acid substitution. In some embodiments, the human ILl5Ra is a mature full-length ILl5Ra. In some embodiments, the pair of affinity domains is selected from the group consisting of: barnase and barnstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. In some embodiments, the first chimeric polypeptide and/or the second chimeric polypeptide further include a signal sequence at its N-terminal end. In some embodiments, the first chimeric polypeptide and/or the second chimeric polypeptide lacks a signal sequence at its N-terminal end. In some embodiments, the first chimeric polypeptide and/or the second chimeric polypeptide further include a signal sequence at its N-terminal end. In some embodiments, the first chimeric polypeptide and/or the second chimeric polypeptide lacks a signal sequence at its N-terminal end.

Also provided herein are methods of increasing the glucose consumption of an immune cell that include: contacting an immune cell in a liquid culture medium including an effective amount of (i) a single-chain chimeric polypeptide including a first target- binding domain, a linker domain, and a second target-binding domain, and optionally (ii) an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for glucose consumption in the immune cell, where the first target-binding domain and the second target-binding domain are each independently selected from the group of: a soluble interleukin, soluble cytokine protein, or soluble cell surface protein, an antigen-binding domain, a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor, and ligands of co-stimulatory molecules.

Also provided herein are methods of increasing the oxidative phosphorylation of an immune cell that include: contacting an immune cell in a liquid culture medium including an effective amount of (i) a single-chain chimeric polypeptide including a first target-binding domain, a linker domain, and a second target-binding domain, and optionally (ii) an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for oxidative phosphorylation in the immune cell, where the first target-binding domain and the second target-binding domain are each independently selected from the group of: a soluble interleukin, soluble cytokine protein, or soluble cell surface protein, an antigen- binding domain, a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor, and ligands of co-stimulatory molecules.

Also provided herein are methods of increasing the aerobic glycolysis of an immune cell that include: contacting an immune cell in a liquid culture medium including an effective amount of (i) a single-chain chimeric polypeptide including a first target- binding domain, a linker domain, and a second target-binding domain, and optionally (ii) an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for aerobic glycolysis in the immune cell, where the first target-binding domain and the second target-binding domain are each independently selected from the group of: a soluble interleukin, soluble cytokine protein, or soluble cell surface protein, an antigen-binding domain, a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor, and ligands of co stimulatory molecules.

Also provided herein are methods of increasing the extracellular acidification rate (ECAR) of an immune cell that include: contacting an immune cell in a liquid culture medium including an effective amount of (i) a single-chain chimeric polypeptide including a first target-binding domain, a linker domain, and a second target-binding domain, and optionally (ii) an IgGl antibody construct that includes at least one antigen binding domain that binds specifically to the linker domain, under conditions that allow for extracellular acidification by the immune cell, where the first target-binding domain and the second target-binding domain are each independently selected from the group of: a soluble interleukin, soluble cytokine protein, or soluble cell surface protein, an antigen binding domain, a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor, and ligands of co-stimulatory molecules.

Also provided herein are methods of increasing the mitochondrial oxygen consumption rate of an immune cell that include: contacting an immune cell in a liquid culture medium including an effective amount of (i) a single-chain chimeric polypeptide including a first target-binding domain, a linker domain, and a second target-binding domain, and optionally (ii) an IgGl antibody construct that includes at least one antigen- binding domain that binds specifically to the linker domain, under conditions that allow for mitochondrial oxygen consumption rate by the immune cell, where the first target- binding domain and the second target-binding domain are each independently selected from the group of: a soluble interleukin, soluble cytokine protein, or soluble cell surface protein, an antigen-binding domain, a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor, and ligands of co-stimulatory molecules.

In some embodiments of any of the methods described herein, the liquid culture medium includes the single-chain chimeric polypeptide and the IgGl antibody construct. In some embodiments of any of the methods described herein, the IgGl antibody construct is a monoclonal IgGl antibody, where both of the antigen-binding domains in the monoclonal IgGl antibody bind specifically to the linker domain. In some embodiments of any of the methods described herein, the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the linker domain.

In some embodiments of any of the methods described herein, the contacting step is performed for a period of about 2 hours to about 20 days (e.g., about 1 day to about 15 days). In some embodiments of any of the methods described herein, the liquid culture medium is a serum-free liquid culture medium. In some embodiments of any of the methods described herein, the liquid culture medium is a chemically-defined liquid culture medium. In some embodiments of any of the methods described herein, the liquid culture medium includes serum. In some embodiments of any of the methods described herein, the liquid culture medium includes the single-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0.5: 1 to about 2: 1 (e.g., about 0.8: 1 to about 1.2: 1).

In some embodiments of any of the methods described herein, the first target binding domain and the linker domain directly abut each other. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the linker domain. In some embodiments of any of the methods described herein, the linker domain and the second target-binding domain directly abut each other. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further includes a linker sequence between the linker domain and the second target-binding domain. In some embodiments of any of the methods described herein, the first target- binding domain and the second target-binding domain directly abut each other.

In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further includes a linker sequence between the first target-binding domain and the second target-binding domain. In some embodiments of any of the methods described herein, the second target-binding domain and the linker domain directly abut each other. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further includes a linker sequence between the second target-binding domain and the linker domain.

In some embodiments of any of the methods described herein, the first target- binding domain and the second target-binding domain bind specifically to the same antigen. In some embodiments of any of the methods described herein, the first target- binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of any of the methods described herein, the first target- binding domain and the second target-binding domain include the same amino acid sequence. In some embodiments of any of the methods described herein, the first target- binding domain and the second target-binding domain bind specifically to different antigens.

In some embodiments of any of the methods described herein, one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain. In some embodiments of any of the methods described herein, the first target- binding domain and the second target-binding domain are each an antigen-binding domain. In some embodiments of any of the methods described herein, the antigen binding domain includes a scFv or a single domain antibody.

In some embodiments of any of the methods described herein, one or both of the first target-binding domain and the second target-binding domain bind to a target selected from the group of: CDl6a, CD28, CD3, CD33, CD20, CD 19, CD22, CD52, CD 123, IL- 1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD 137, CD40,

CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16- binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF-b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-l 2, a receptor for IL-l 5, a receptor for IL-l 7, a receptor for IL-l 8, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.

In some embodiments of any of the methods described herein, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. In some embodiments of any of the methods described herein, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-l 2, IL-15, IL-l 7, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

In some embodiments of any of the methods described herein, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor. In some

embodiments of any of the methods described herein, the soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28.

In some embodiments of any of the methods described herein, the linker domain is a soluble tissue factor domain. In some embodiments of any of the methods described herein, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments of any of the methods described herein, the soluble human tissue factor domain comprises a sequence that is at least 80% identical, at least 90% identical, or at least 95% identical to SEQ ID NO: 1. In some embodiments of any of the methods described herein, the soluble human tissue factor domain does not include one or more of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

In some embodiments of any of the methods described herein, the soluble human tissue factor domain does not include any of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

In some embodiments of any of the methods described herein, the soluble tissue factor domain is not capable of binding Factor Vila. In some embodiments of any of the methods described herein, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide does not stimulate blood coagulation in a mammal. In some embodiments of any of the methods described herein, the IgGl antibody construct includes at least one antigen-binding domain that binds specifically to the soluble tissue factor domain. In some embodiments of any of the methods described herein, the linker domain is selected from the group of: a kappa chain and a lambda chain. In some embodiments of any of the methods described herein, the IgGl antibody construct is a monoclonal IgGl antibody, where both of the antigen-binding domains in the monoclonal IgGl antibody bind specifically to the linker domain. In some embodiments of any of the methods described herein, the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the linker domain.

In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further includes one or more additional target-binding domains at its N- and/or C-terminus. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide includes one or more additional target-binding domains at its N-terminus. In some embodiments of any of the methods described herein, one or more additional target-binding domains directly abuts the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further includes a linker sequence between one of the at least one additional target-binding domains and the first target-binding domain, the second target-binding domain, or the linker domain.

In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide includes one or more additional target-binding domains at its C- terminus. In some embodiments of any of the methods described herein, one of the one or more additional target-binding domains directly abuts the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further includes a linker sequence between one of the at least one additional target-binding domains and the first target-binding domain, the second target-binding domain, or the linker domain.

In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide includes one or more additional target binding domains at its N- terminus and the C-terminus. In some embodiments of any of the methods described herein, one of the one or more additional target-binding domains at the N-terminus directly abuts the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments of any of the methods described herein, the single- chain chimeric polypeptide further includes a linker sequence between one of the one or more additional target-binding domains at the N-terminus and the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments of any of the methods described herein, one of the one or more additional target-binding domains at the C-terminus directly abuts the first target-binding domain, the second target-binding domain, or the linker domain. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further includes a linker sequence between one of the one or more additional target-binding domains at the C- terminus and the first target-binding domain, the second target-binding domain, or the linker domain.

In some embodiments of any of the methods described herein, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments of any of the methods described herein, two or more of the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains bind specifically to the same epitope. In some embodiments of any of the methods described herein, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains include the same amino acid sequence. In some embodiments of any of the methods described herein, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same antigen. In some embodiments of any of the methods described herein, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same epitope. In some embodiments of any of the methods described herein, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each include the same amino acid sequence. In some embodiments of any of the methods described herein, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to different antigens.

In some embodiments of any of the methods described herein, one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen-binding domain. In some embodiments of any of the methods described herein, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen binding domain. In some embodiments of any of the methods described herein, the antigen-binding domain includes a scFv or a single domain antibody.

In some embodiments of any of the methods described herein, one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL- 10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4,

TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF-b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL- 3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-l 2, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a LILP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD28.

In some embodiments of any of the methods described herein, one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. In some embodiments of any of the methods described herein, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL- 18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

In some embodiments of any of the methods described herein, one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor. In some embodiments of any of the methods described herein, the soluble interleukin, soluble cytokine receptor, or soluble cell surface receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28.

In some embodiments of any of the methods described herein, one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a ligand of a co-stimulatory molecule. In some embodiments of any of the methods described herein, the ligand of a co- stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l.

In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further includes a peptide tag positioned at the N-terminal end or the C-terminal end of the single-chain chimeric polypeptide. In some embodiments of any of the methods described herein, the immune cell was previously obtained from a subject. Some embodiments of any of the methods described herein further include obtaining the immune cell from the subject prior to the contacting step. In some embodiments of any of the methods described herein, the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor. Some embodiments of any of the methods described herein further include, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Some embodiments of any of the methods described herein further include, before the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a

recombinant T-cell receptor. Some embodiments of any of the methods described herein further include, after the contacting step, isolating the immune cell. In some

embodiments of any of the methods described herein, after the contacting step, the immune cell has an increased level of expression or secretion of one or more proteins selected from the group of: TNF-a, IFN-T, granzyme A, granzyme B, perforin, 2B4,

CD8, CDl la, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, CDl58a, CDl58b, CDl58e, CD178, CD226, CD253, NKG2A, NKG2C, NKG2D, LIR-l, LILR-B1, KIR2DL1, KIR3DL1, KIR2DL2, KIR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-l, NKG2A, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-a, and IFN-g as compared to the level of expression or secretion of the one or more proteins prior to the contacting step administering the immune cell to a subject in need thereof.

In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having an age-related disease or condition. In some embodiments of any of the methods described herein, the age-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having a cancer. In some embodiments of any of the methods described herein, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some embodiments of any of the methods described herein, the subject has been diagnosed or identified as having an infectious disease. In some embodiments of any of the methods described herein, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, or influenza virus.

Also provided herein are activated immune cells produced by any of the methods described herein. Also provided herein are pharmaceutical compositions that include any of the activated immune cells produced by any of the methods described herein. Also provided herein are kits that include a pharmaceutical composition including any of the activated immune cells described herein produced by any of the methods described herein.

Also provided herein are methods of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced by any of the methods described herein or any of the pharmaceutical compositions described herein. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having a cancer. In some embodiments of any of the methods described herein, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having an aging-related disease or condition. In some embodiments of any of the methods described herein, the aging-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease,

spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Also provided herein are methods of treating a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced by any of the methods described herein or any of the pharmaceutical compositions described herein. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having a cancer. In some embodiments of any of the methods described herein, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non- Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having an aging-related disease or condition. In some embodiments of any of the methods described herein, the aging-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease,

spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some embodiments of any of the methods described herein, the subject has been diagnosed or identified as having an infectious disease. In some embodiments of any of the methods described herein, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, or influenza virus.

Also provided herein are methods of increasing the glucose consumption of an immune cell that include: contacting an immune cell in a liquid culture medium including (1) an effective amount of a multi-chain chimeric polypeptide including: (a) a first chimeric polypeptide including: (i) a first target-binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target binding domain, where the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgGl antibody construct including at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for glucose consumption in the immune cell.

Also provided herein are methods of increasing the oxidative phosphorylation of an immune cell that include: contacting an immune cell in a liquid culture medium including (1) an effective amount of a multi-chain chimeric polypeptide including: (a) a first chimeric polypeptide including: (i) a first target-binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, where the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgGl antibody construct including at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for oxidative phosphorylation in the immune cell.

Also provided herein are methods of increasing the aerobic glycolysis of an immune cell that include: contacting an immune cell in a liquid culture medium including (1) an effective amount of a multi-chain chimeric polypeptide including: (a) a first chimeric polypeptide including: (i) a first target-binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target binding domain, where the first chimeric polypeptide and the second chimeric

polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgGl antibody construct including at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for aerobic glycolysis in the immune cell.

Also provided herein are methods of increasing the extracellular acidification rate (ECAR) of an immune cell that include: contacting an immune cell in a liquid culture medium including (1) an effective amount of a multi-chain chimeric polypeptide including: (a) a first chimeric polypeptide including: (i) a first target-binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, where the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgGl antibody construct including at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for extracellular acidification by the immune cell.

Also provided herein are methods of increasing the mitochondrial oxygen consumption rate of an immune cell that include: contacting an immune cell in a liquid culture medium including (1) an effective amount of a multi-chain chimeric polypeptide including: (a) a first chimeric polypeptide including: (i) a first target-binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, where the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgGl antibody construct including at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for mitochondrial oxygen consumption rate by the immune cell.

In some embodiments of any of the methods described herein, the liquid culture medium includes the single-chain chimeric polypeptide and the IgGl antibody construct. In some embodiments of any of the methods described herein, the IgGl antibody construct is a monoclonal IgGl antibody, where both of the antigen-binding domains in the monoclonal IgGl antibody bind specifically to the linker domain. In some embodiments of any of the methods described herein, the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the linker domain. In some embodiments of any of the methods described herein, the contacting step is performed for a period of about 2 hours to about 20 days (e.g., about 1 day to about 15 days).

In some embodiments of any of the methods described herein, the liquid culture medium is a serum-free liquid culture medium. In some embodiments of any of the methods described herein, the liquid culture medium is a chemically-defined liquid culture medium. In some embodiments of any of the methods described herein, the liquid culture medium includes serum. In some embodiments of any of the methods described herein, the liquid culture medium includes the single-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0 5: 1 to about 2: 1 (e.g., about 0 8: 1 to about 1.2: 1).

In some embodiments of any of the methods described herein, the first target- binding domain and the linker domain directly abut each other in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further includes a linker sequence between the first target-binding domain and the linker domain in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the linker domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between the linker domain and the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric

polypeptide. In some embodiments of any of the methods described herein, the second chimeric polypeptide further includes a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of any of the methods described herein, the first target- binding domain and the second target-binding domain bind specifically to the same antigen. In some embodiments of any of the methods described herein, the first target- binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of any of the methods described herein, the first target- binding domain and the second target-binding domain include the same amino acid sequence. In some embodiments of any of the methods described herein, the first target- binding domain and the second target-binding domain bind specifically to different antigens. In some embodiments of any of the methods described herein, one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain. In some embodiments of any of the methods described herein, the first target- binding domain and the second target-binding domain are each antigen-binding domains. In some embodiments of any of the methods described herein, the antigen-binding domain comprises a scFv or a single domain antibody. In some embodiments of any of the methods described herein, one or both of the first target-binding domain and the second target-binding domain bind specifically to a target selected from the group of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa,

CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD 137, CD40, CD47, CD70, 0X40, IGF- 1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF-b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL- 3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-l 2, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a EILP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD28.

In some embodiments of any of the methods described herein, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. In some embodiments of any of the methods described herein, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL- 12, IL-l 5, IL-l 7, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16- binding protein. In some embodiments of any of the methods described herein, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor. In some

embodiments of any of the methods described herein, the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble

NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28.

In some embodiments of any of the methods described herein, the first chimeric polypeptide further includes one or more additional target-binding domain(s), where at least one of the one or more target-binding domain(s) is positioned between the linker domain and the first domain of the pair of affinity domains. In some embodiments of any of the methods described herein, the first chimeric polypeptide further includes a linker sequence between the linker domain and the at least one of the one or more target antigen-binding domain(s), and/or a linker sequence between the at least one of the one or more target antigen-binding domain(s) and the first domain of the pair of affinity domains.

In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises one or more additional target-binding domains at the N- terminal and/or C-terminal end of the first chimeric polypeptide. In some embodiments of any of the methods described herein, at least one of the one or more additional target binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further includes a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains. In some embodiments of any of the methods described herein, the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further includes a linker sequence between the at least one of the one or more additional target-binding domains and the first target-binding domain. In some embodiments of any of the methods described herein, at least one of the one or more additional target-binding domains is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains is positioned between the linker domain and the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further includes a linker sequence disposed between the at least one additional target- binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further includes a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains, directly abuts the linker domain and/or the first domain of the pair of affinity domains. In some embodiments of any of the methods described herein, the first chimeric polypeptide further includes a linker sequence disposed (i) between the linker domain and the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains.

In some embodiments of any of the methods described herein, the second chimeric polypeptide further includes one or more additional target-binding domains at the N-terminal end and/or the C-terminal end of the second chimeric polypeptide. In some embodiments of any of the methods described herein, at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide. In some embodiments of any of the methods described herein, the second chimeric polypeptide further includes a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide. In some embodiments of any of the methods described herein, at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide. In some embodiments of any of the methods described herein, the second chimeric polypeptide further includes a linker sequence between at least one of the one or more additional target-binding domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of any of the methods described herein, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments of any of the methods described herein, two or more of the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains bind specifically to the same epitope. In some embodiments of any of the methods described herein, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains include the same amino acid sequence. In some embodiments of any of the methods described herein, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same antigen. In some embodiments of any of the methods described herein, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same epitope. In some embodiments of any of the methods described herein, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each include the same amino acid sequence. In some embodiments of any of the methods described herein, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to different antigens. In some embodiments of any of the methods described herein, one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen-binding domain. In some embodiments of any of the methods described herein, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen binding domain. In some embodiments of any of the methods described herein, the antigen-binding domain comprises a scFv.

In some embodiments of any of the methods described herein, one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL- 10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF-b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL- 3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-l 2, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a LILP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD3, and a receptor for CD28.

In some embodiments of any of the methods described herein, one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. In some embodiments of any of the methods described herein, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL- 18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

In some embodiments of any of the methods described herein, one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor. In some embodiments of any of the methods described herein, the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD 122, a soluble CD3, or a soluble CD28.

In some embodiments of any of the methods described herein, the first chimeric polypeptide further includes a peptide tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide. In some embodiments of any of the methods described herein, the second chimeric polypeptide further includes a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of any of the methods described herein, the linker domain is a soluble tissue factor domain. In some embodiments of any of the methods described herein, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments of any of the methods described herein, the soluble human tissue factor domain includes a sequence that is at least 80% identical, at least 90% identical, or at least 95% identical to SEQ ID NO: 1. In some embodiments of any of the methods described herein, the soluble human tissue factor domain does not include one or more of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. In some embodiments of any of the methods described herein, the soluble human tissue factor domain does not include any of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. In some embodiments of any of the methods described herein, the soluble tissue factor domain is not capable of binding to Factor Vila. In some embodiments of any of the methods described herein, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments of any of the methods described herein, the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal. In some embodiments of any of the methods described herein, the IgGl antibody construct includes at least one antigen-binding domain that binds specifically to the soluble tissue factor domain. In some embodiments of any of the methods described herein, the linker domain is selected from the group of: a kappa chain and a lambda chain. In some embodiments of any of the methods described herein, the IgGl antibody construct is a monoclonal IgGl antibody, where both antigen-binding domains in the monoclonal IgGl antibody bind specifically to the linker domain. In some embodiments of any of the methods described herein, the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the linker domain.

In some embodiments of any of the methods described herein, the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (ILl5Ra) and a soluble IL-15. In some embodiments of any of the methods described herein, the soluble IL15 has a D8N or D8A amino acid substitution. In some embodiments of any of the methods described herein, the human ILl5Ra is a mature full-length ILl5Ra. In some embodiments of any of the methods described herein, the pair of affinity domains is selected from the group consisting of: bamase and barnstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

In some embodiments of any of the methods described herein, the first chimeric polypeptide and/or the second chimeric polypeptide further includes a signal sequence at its N-terminal end. In some embodiments of any of the methods described herein, the first chimeric polypeptide and/or the second chimeric polypeptide lacks a signal sequence at its N-terminal end.

In some embodiments of any of the methods described herein, the immune cell was previously obtained from a subject. Some embodiments of any of the methods described herein further include obtaining the immune cell from the subject prior to the contacting step. In some embodiments of any of the methods described herein, the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor. Some embodiments of any of the methods described herein further include, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Some embodiments of any of the methods described herein further include, before the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Some embodiments of any of the methods described herein further include, after the contacting step, isolating the immune cell. In some embodiments of any of the methods described herein, after the contacting step, the immune cell has an increased level of expression or secretion of one or more proteins selected from the group of: TNF-a, IFN-Y, granzyme A, granzyme B, perforin, 2B4, CD8, CDl la, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, CDl58a, CDl58b, CDl58e, CD178, CD226, CD253,

NKG2A, NKG2C, NKG2D, LIR-l, LILR-B1, KIR2DL1, KIR3DL1, KIR2DL2, KIR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-l, NKG2A, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-a, and IFN-g as compared to the level of expression or secretion of the one or more proteins prior to the contacting step. Some embodiments of any of the methods described herein further include, after the contacting step, administering the immune cell to a subject in need thereof.

In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having an age-related disease or condition. In some embodiments of any of the methods described herein, the age-related disease or condition is selected from the consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having a cancer. In some embodiments of any of the methods described herein, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments of any of the methods described herein, the subject has been diagnosed or identified as having an infectious disease. In some embodiments of any of the methods described herein, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, or influenza virus.

Also provided herein are activated immune cells produced using any of the methods described herein. Also pharmaceutical compositions that include any of the activated immune cells described herein produced by any of the methods described herein. Also provided herein are kits that include any of the pharmaceutical compositions described herein that include any of the activated immune cells described herein produced by any of the methods described herein.

Also provided herein are methods of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced using any of the methods described herein or any of the pharmaceutical compositions described herein.

In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having a cancer. In some embodiments of any of the methods described herein, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having an aging-related disease or condition. In some embodiments of any of the methods described herein, the aging-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease,

spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Also provided herein are methods of treating a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced by any of the methods described herein or any of the pharmaceutical compositions described herein. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having a cancer. In some embodiments of any of the methods described herein, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non- Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having an aging-related disease or condition. In some embodiments of any of the methods described herein, the aging-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis,

inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease,

spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some embodiments of any of the methods described herein, the subject has been diagnosed or identified as having an infectious disease. In some embodiments of any of the methods described herein, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, or influenza virus.

In some embodiments of any of the methods or kits described herein, the soluble human tissue factor domain does not stimulate blood coagulation. In some embodiments of any of the methods or kits described herein, the soluble tissue factor domain comprises or consists of a sequence from a wildtype soluble human tissue factor (or any sequence therefrom).

Also provided herein are methods of inducing differentiation of an immune cell into a memory or memory -like immune cell that include contacting an immune cell in a liquid culture medium including: (1) an effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide including: (i) a first target binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, where the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgGl antibody construct including at least one antigen-binding domain that binds specifically to the linker domain.

Also provided herein are methods of inducing differentiation of an immune cell into a memory or memory -like immune cell that include contacting an immune cell in a liquid culture medium including an effective amount of (i) a single-chain chimeric polypeptide including a first target-binding domain, a linker domain, and a second target binding domain, and (ii) an IgGl antibody construct that includes at least one antigen- binding domain that binds specifically to the linker domain.

In some embodiments of any of the methods described herein, the immune cell was previously obtained from a subject. In some embodiments of any of the methods described herein, the immune cell is selected from the group c of: an immature thymocyte, a periperhal blood Treg cell, a Thl7 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, ld T cell, an ab T cell, a tumor-infiltrating T cell, a CD8 ⁺ T cell, a CD4 ⁺ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell. In some embodiments of any of the methods described herein, the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

Some embodiments of any of the methods described herein further include, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-recpetor or a recombinant T-cell receptor. Some embodiments of any of the methods described herein further include administering to the immune cell to a subject in need thereof.

As used herein, the term“chimeric” refers to a polypeptide that includes amino acid sequences (e.g., domains) originally derived from two different sources (e.g., two different naturally-occurring proteins, e.g., from the same or different species). For example, a chimeric polypeptide can include domains from at least two different naturally occurring human proteins. In some examples, a chimeric polypeptide can include a domain that is a synthetic sequence (e.g., a scFv) and a domain that is derived from a naturally-occurring protein (e.g., a naturally-occurring human protein). In some embodiments, a chimeric polypeptide can include at least two different domains that are synthetic sequences (e.g., two different scFvs).

An“antigen-binding domain” is one or more protein domain(s) (e.g., formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides) that is capable of specifically binding to one or more different antigen(s). In some examples, an antigen-binding domain can bind to an antigen or epitope with specificity and affinity similar to that of naturally-occurring antibodies. In some embodiments, the antigen-binding domain can be an antibody or a fragment thereof. In some embodiments, an antigen-binding domain can include an alternative scaffold. Non-limiting examples of antigen-binding domains are described herein. Additional examples of antigen-binding domains are known in the art.

A“soluble tissue factor domain” refers to a polypeptide having at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical) to a segment of a wildtype mammalian tissue factor protein (e.g., a wildtype human tissue factor protein) that lacks the transmembrane domain and the intracellular domain. Non-limiting examples of soluble tissue factor domains are described herein.

The term“soluble interleukin protein” is used herein to refer to a mature and secreted interleukin protein or a biologically active fragment thereof. In some examples, a soluble interleukin protein can include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a wildtype mature and secreted mammalian interleukin protein (e.g., a wildtype human interleukin protein) and retains its biological activity. Non-limiting examples of soluble interleukin proteins are described herein.

The term“soluble cytokine protein” is used herein to refer to a mature and secreted cytokine protein or a biologically active fragment thereof. In some examples, a soluble cytokine protein can include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a wildtype mature and secreted mammalian interleukin protein (e.g., a wildtype human interleukin protein) and retains its biological activity. Non-limiting examples of soluble cytokine proteins are described herein.

The term“soluble interleukin receptor” is used herein in the broadest sense to refer to a polypeptide that lacks a transmembrane domain (and optionally an intracellular domain) that is capable of binding one or more of its natural ligands (e.g., under physiological conditions, e.g., in phosphate buffered saline at room temperature). For example, a soluble interleukin receptor can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to an extracellular domain of wildtype interleukin receptor and retains its ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally, further lacks its intracellular domain). Non-limiting examples of soluble interleukin receptors are described herein.

The term“soluble cytokine receptor” is used herein in the broadest sense to refer to a polypeptide that lacks a transmembrane domain (and optionally an intracellular domain) that is capable of binding one or more of its natural ligands (e.g., under physiological conditions, e.g., in phosphate buffered saline at room temperature). For example, a soluble cytokine receptor can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to an extracellular domain of wildtype cytokine receptor and retains its ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally, further lacks its intracellular domain). Non-limiting examples of soluble cytokine receptors are described herein.

The term“ligand of a co- stimulatory molecule” is used herein in the broadest sense to refer to a polypeptide that is capable of binding and activating a co-stimulatory receptor molecule on an immune cell (e.g., under physiological conditions, e.g., in phosphate buffered saline at room temperature). For example, a ligand of a co stimulatory molecule can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to a ligand of a co- stimulatory molecule that retains its ability to specifically bind to one or more of its natural receptors. Non-limiting examples of ligands of co-stimulatory molecules are described herein.

The term“antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules that include one or more antigen-binding domains that specifically bind to an antigen or epitope. An antibody specifically includes, e.g., intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multi-specific antibodies. One example of an antigen-binding domain is an antigen-binding domain formed by a VH -VL dimer. Additional examples of an antibody are described herein. Additional examples of an antibody are known in the art.

“Affinity” refers to the strength of the sum total of non-covalent interactions between an antigen-binding site and its binding partner (e.g., an antigen or epitope). Unless indicated otherwise, as used herein,“affinity” refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of an antigen-binding domain and an antigen or epitope. The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein.

Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

Additional methods for determining the affinity for an antigen-binding domain and its corresponding antigen or epitope are known in the art.

A“single-chain polypeptide” as used herein to refers to a single protein chain. The term“pair of affinity domains” is two different protein domain(s) that bind specifically to each other with a KD of less than of less than 1 x 10 ⁷ M (e.g., less than 1 x 10 ⁸ M, less than 1 x 10 ⁹ M, less than 1 x 10 ¹⁰ M, or less than 1 x 10 ¹¹ M). In some examples, a pair of affinity domains can be a pair of naturally-occurring proteins. In some embodiments, a pair of affinity domains can be a pair of synthetic proteins. Non- limiting examples of pairs of affinity domains are described herein. The term“epitope” means a portion of an antigen that specifically binds to an antigen-binding domain. Epitopes can, e.g., consist of surface-accessible amino acid residues and/or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non- conformational epitopes are distinguished in that the binding to the former but not the latter may be lost in the presence of denaturing solvents. An epitope may comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. Methods for identifying an epitope to which an antigen-binding domain binds are known in the art.

An“immune effector cell” refers to a cell of the immune system of a mammal that is capable, directly or indirectly, of recognizing and/or causing cytostasis or cell death of a pathogenic cell (e.g., a cancer cell) in the mammal. Non-limiting examples of immune effector cells include macrophages, natural killer cells, T-lymphocytes (e.g., cytotoxic T-lymphocytes and T-helper cells), neutrophils, monocytes, and eosinophils. Additional examples of immune effector cells are known in the art.

The term“treatment” means to ameliorate at least one symptom of a disorder. In some examples, the disorder being treated is cancer and to ameliorate at least one symptom of cancer includes reducing aberrant proliferation, gene expression, signaling, translation, and/or secretion of factors. Generally, the methods of treatment include administering a therapeutically effective amount of composition that reduces at least one symptom of a disorder to a subject who is in need of, or who has been determined to be in need of such treatment.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting.

All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows a schematic diagram of an exemplary IL-7/IL-l5RaSu DNA construct.

Figure 2 shows a schematic diagram of an exemplary IL-21/TF/IL-15 DNA construct.

Figure 3 shows a schematic diagram of the interaction between the exemplary IL- 7/IL-l5RaSu and IL-21/TF/IL-15 DNA constructs.

Figure 4 shows a schematic diagram of the interaction between the exemplary IL-

7/IL-l5RaSu and IL-21/TF/IL-15 fusion proteins resulting in an IL-2l/TF/IL-l5:IL-7/IL- l5RaSu complex (2ltl5-7s).

Figure 5 shows the expansion of primary natural killer (NK) cells by stimulation with 2ltl5-7s + anti-TF IgGl antibody.

Figure 6 shows activation of expanded primary NK cells, using CD25 MFI and

CD69 MFI as markers of NK cell activation.

Figure 7 shows cytotoxic activity of expanded NK cells against K562 human tumor cells, wherein NK cells stimulated with 2ltl5-7s + anti-TF IgGl antibody demonstrate greater specific lysis of K562 cells than NK cells not stimulated with 2ltl 5- 7s + anti-TF IgGl antibody.

Figure 8 shows a schematic diagram of an exemplary TGF-PRII/IL- 15RaSu DNA construct.

Figure 9 shows a schematic diagram of an exemplary IL-21/TF/IL-15 DNA construct.

Figure 10 shows a schematic diagram of the interaction between the exemplary

TGF-pRIML-l5RaSu and IL-21/TF/IL-15 DNA constructs.

Figure 11 shows a schematic diagram of the interaction between the exemplary TGF-PRII /IL-l5RaSu and IL-21/TF/IL-15 fusion proteins resulting in an IL-21/TF/IL- 15: TGF-pRII/IL-l5RaSU complex (2ltl5-TGFRs). Figure 12 shows the expansion of primary natural killer (NK) cells by stimulation with 2ltl5-TGFRs + anti-TF IgGl antibody (dark line with squares) or primary NK cells contacted with 2ltl5-TGFRs alone, anti-TF IgGl antibody, anti-TF IgG4 antibody, or with a combination of 2ltl5-TGFRs + anti-TF IgG4 antibody (all other data shown).

Figure 13 shows activation of expanded primary NK cells, using CD25 MFI (top) and CD69 MFI (bottom) as markers of NK cell activation.

Figure 14 shows a schematic diagram of an exemplary IL-2l/IL-l5RaSu DNA construct.

Figure 15 shows a schematic diagram of an exemplary IL-7/TF/IL-15 DNA construct.

Figure 16 shows a schematic diagram of the interaction between the exemplary IL-2l/IL-l5RaSu and IL-7/TF/IL-15 DNA constructs.

Figure 17 shows a schematic diagram of the interaction between the exemplary IL-2l/IL-l5RaSu and IL-7/TF/IL-15 fusion proteins resulting in an IL-7/TF/IL-15TL- 2l/IL-l5RaSu complex (7tl 5-21 s).

Figure 18 shows in diagrammatic form the activation and expansion of primary natural killer (NK) cells by stimulation with 2ltl5-TGFRs + anti-TF IgGl antibody.

Figure 19 shows size exclusion chromatography (SEC) profiles of anti-TF IgGl antibody, 7tl 5-21 s and the complex containing equal amounts of anti-TF IgGl antibody and 7tl5-2ls.

Figure 20 is a graph showing the oxygen consumption rate (OCR) in pmoles/min for human NK cells isolated from blood (1 x 10 ⁶ cells/mL) and stimulated with (1) 50 nM anti-TF IgGl (IgGl), (2) 100 nM 7tl5-2ls and 50 nM anti-TF IgGl, or (3) 100 nM 2 ltl 5-7s and 50 nM anti-TF IgGl for up to 5 days at 37 °C, 5% C0 ₂ in RPMI 1640 supplemented with 4 mM L-glutamine, penicillin, streptomycin, non-essential amino acids, sodium pyruvate, and 10% fetal bovine serum. Cells were maintained at a concentration of 0.5 x 10 ⁶ cells/mL to 2.0 x 10 ⁶ cells/mL until day 5. Glycolysis stress tests were performed in Seahorse media containing 2 mM glutamine. The drug concentrations used during the assay were as follows: 10 mM glucose, 100 nm oligomycin, and 100 mM 2-deoxy-D-glucose. Figure 21 is a graph showing the extracellular acidification rate (ECAR) in mpH/minute for human NK cells isolated from blood (1 x 10 ⁶ cells/mL) and stimulated with (1) 50 nM anti-TF IgGl (IgGl), (2) 100 nM 7tl5-2ls and 50 nM anti-TF IgGl, or (3) 100 nM 2 ltl 5-7s and 50 nM anti-TF IgGl for up to 5 days at 37 °C, 5% CO2 in RPMI 1640 supplemented with 4 mM L-glutamine, penicillin, streptomycin, non- essential amino acids, sodium pyruvate, and 10% fetal bovine serum. Cells were maintained at a concentration of 0.5 x 10 ⁶ cells/mL to 2.0 x 10 ⁶ cells/mL until day 5. Glycolysis stress tests were performed in Seahorse media containing 2 mM glutamine. The drug concentrations used during the assay were as follows: 10 mM glucose, 100 nm oligomycin, and 100 mM 2-deoxy-D-glucose.

Figure 22 is a schematic showing the structure of the l8tl5-l2s construct.

Figure 23 are two graphs showing the oxygen consumption rate (OCR) in pmoles/min for human NK cells isolated from blood (2 x 10 ⁶ cells/mL) of two different donors. The isolated NK cells were left unstimulated or were stimulated with (1) 100 nM l8tl5-l2s (“1812”) or a mixture of (2) recombinant human IL-12 (0.25 pg), recombinant human IL-15 (1.25 pg), and recombinant human IL-18 (1.25 pg) (“single cytokines”), overnight at 37 °C, 5% CO2 in RPMI 1640 supplemented with 4 mM L-glutamine, penicillin, streptomycin, non-essential amino acids, sodium pyruvate, and 10% fetal bovine serum. On the next day, the cells were harvested and extracellular flux assays on expanded NK cells were performed using a XFp Analyzer (Seahorse Bioscience). The harvested cells were washed and plated at 2.0 x 10 ⁵ cells/well in at least duplicate for extracellular flux analysis of oxygen consumption rate. Glycolysis stress tests were performed in Seahorse Media contain 2 mM glutamine. Drug concentrations during the assay were: 10 mM glucose, 100 nM oligomycin, and 100 mM 2-deoxy-D-glucose.

Figure 24 are two graphs showing the extracellular acidification rate (ECAR) in mpH/minute for human NK cells isolated from blood (2 x 10 ⁶ cells/mL) of two different donors. The isolated NK cells were left unstimulated or were stimulated with (1) 100 nM l8tl5-l2s (“1812”) or a mixture of (2) recombinant human IL-12 (0.25 pg), recombinant human IL-15 (1.25 pg), and recombinant human IL-18 (1.25 pg) (“single cytokines”), overnight at 37 °C, 5% CO2 in RPMI 1640 supplemented with 4 mM L- glutamine, penicillin, streptomycin, non-essential amino acids, sodium pyruvate, and 10% fetal bovine serum. On the next day, the cells were harvested and extracellular flux assays on expanded NK cells were performed using a XFp Analyzer (Seahorse

Bioscience). The harvested cells were washed and plated at 2.0 x 10 ⁶ cells/well in at least duplicate for extracellular flux analysis of extracellular flux analysis (ECAR). Glycolysis stress tests were performed in Seahorse Media contain 2 mM glutamine. Drug concentrations during the assay were: 10 mM glucose, 100 nM oligomycin, and 100 mM 2-deoxy-D-glucose.

Figure 25 shows a schematic diagram of an exemplary IL-l2/IL-l5RaSu DNA construct.

Figure 26 shows a schematic diagram of an exemplary IL-18/TF/IL-15 DNA construct.

Figure 27 shows a schematic diagram of the interaction between the exemplary IL-l2/IL-l5RaSu and IL-18/TF/IL-15 DNA constructs.

Figure 28 shows a schematic diagram of the interaction between the exemplary IL-l2/IL-l5RaSu and IL-18/TF/IL-15 fusion proteins resulting in IL-l8/TF/IL-l5:IL- l2/IL-l5RaSu complex (18tl 5-l2s).

Figure 29 shows a chromatograph of l8tl5-l2s purification elution from an anti- TF affinity column.

Figure 30 shows an exemplary chromatographic profile of anti-TF antibody/SEC- purified 18tl 5-l2s protein following elution on an analytical size exclusion column, demonstrating separation of monomeric multiprotein l8tl5-l2s complexes from protein aggregates.

Figure 31 shows an example of a 4-12% SDS-PAGE of the l8tl5-l2s complex following disulfide bond reduction. Lane 1 : SeeBlue Plus2 marker; Lane 2: anti-TF antibody-purified l8tl5-l2s (0.5 pg); Lane 3: anti-TF antibody-purified l8tl5-l2s (1 rig)·

Figure 32 shows SDS PAGE analysis of deglycosylated and non-deglycosylated l8tl5-l2s. Lane 1 : anti-TF antibody-purified l8tl5-l2s (0.5 pg), non-deglycosylated; Lane 2: anti-TF antibody-purified 18tl 5-l2s (1 pg), non-deglycosylated; Lane 3: 18tl 5- l2s (1 pg), deglycosylated, Lane 4: Markl2 unstained maker. Figure 33 shows a sandwich ELISA for the 18tl 5-l2s complex, comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-12 detection antibody (BAF 219).

Figure 34 shows a sandwich ELISA for the 18tl 5-l2s complex, comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-15 detection antibody (BAM 247).

Figure 35 shows a sandwich ELISA for the 18tl 5-l2s complex, comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-18 detection antibody (D045-6).

Figure 36 shows a sandwich ELISA for the 18tl 5-l2s complex, comprising an anti-human tissue factor (143) capture antibody and an anti-human tissue factor detection antibody.

Figure 37 shows proliferation of IL- 15 -dependent 32ϋb cells mediated by the l8tl5-l2s complex (open squares) and recombinant IL-15 (black squares).

Figure 38 shows biological activity of IL-18 within the l8tl5-l2s complex (open squares), where recombinant IL-18 (black squares) and recombinant IL-12 (black circles) serve as positive and negative controls, respectively.

Figure 39 shows biological activity of IL-12 within the l8tl5-l2s complex (open squares), where recombinant IL-12 (black circles) and recombinant IL-18 (open squares) serve as positive and negative controls, respectively.

Figure 40A shows a flow cytometry graph of cell-surface CD25 expression of NK cells induced by the l8tl5-l2s complex. Figure 40B shows a flow cytometry graph of cell-surface CD69 expression of NK cells induced by the l8tl5-l2s complex.

Figure 41 shows a flow cytometry graph of intracellular interferon gamma expression of NK cells induced by the l8tl5-l2s complex.

Figure 42 shows a bar graph showing cytotoxicity of l8tl5-l2s induced human NK cells against K562. K562 cells (labeled with Celltrace violet) at 1 x l0 ^A 5/well and human NK cells (NK cells were isolated from human blood huffy coat with StemCell human NK cell purification kit) at 2 x l0 ^A 5/well were incubated with l8tl5-l2s in RPMI-10 for 20 hours. Cell mixtures were harvested, stained with propidium iodide (PI), and analyzed using a BD FACSCelesta™ flow cytometer. The graph represents duplicated samples.

Figure 43 shows a schematic diagram of an exemplary IL-l2/IL-l5RaSu/aCDl6 DNA construct.

Figure 44 shows a schematic diagram of an exemplary IL-18/TF/IL-15 DNA construct.

Figure 45 shows a schematic diagram of the interaction between the exemplary IL- 12/IL- 15RaSu/aCD 16scFv and IL-18/TF/IL-15 DNA constructs.

Figure 46 shows a schematic diagram of an exemplary l8tl5-l2s/aCDl6 protein complex.

Figure 47 shows a sandwich ELISA for the l8tl5-l2sl6 complex, comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-12 (dark line) or an anti-human tissue factor detection antibody (light line).

Figure 48 shows a schematic diagram of an exemplary TGF RII/IL- 15RaSu DNA construct.

Figure 49 shows a schematic diagram of an exemplary IL-21/TF/IL-15 construct.

Figure 50 shows a schematic diagram of the interaction between the exemplary IL- IL-21/TF/IL-15 and TGF RII/IL- l 5RaSu constructs.

Figure 51 shows a schematic diagram of the interaction between the exemplary TGF RII/IL- l 5RaSu and IL-21/TF/IL-15 fusion proteins, resulting in an IL-21/TF/IL- l5/TGF RII/IL-l5RaSu complex (2ltl5-TGFRs).

Figure 52 shows a chromatograph of 2ltl5-TGFRs purification elution from an anti-TF antibody affinity column.

Figure 53 shows an exemplary 2ltl5-TGFRs size exclusion chromatograph showing a main protein peak and a high molecular weight peak

Figure 54 shows an example of a 4-12% SDS-PAGE of the 2ltl5-TGFRs complex following disulfide bond reduction. Lane 1 : Markl2 unstained marker (numbers on the left side indicate molecular weights in kDa); Lane 2: 2ltl5-TGFRs (0.5 pg); Lane 3: 2ltl5-TGFRs (1 pg); Lane 4: 2ltl5-TGFRs, deglycosylated (1 pg), wherein the MW was the expected size of 53kDa and 39.08 kDa. Figure 55 shows a sandwich ELISA for the 2ltl5-TGFRs complex, comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-21 detection antibody (13-7218-81, BioLegend).

Figure 56 shows a sandwich ELISA for the 2ltl5-TGFRs complex, comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-15 detection antibody (BAM 247, R&D Systems).

Figure 57 shows a sandwich ELISA for the 2ltl5-TGFRs complex, comprising an anti-human tissue factor capture antibody and a biotinylated anti-human TGF RII detection antibody (BAF241, R&D Systems).

Figure 58 shows a sandwich ELISA for the 2ltl5-TGFRs complex, comprising an anti-human tissue factor (143) capture antibody and an anti-human tissue factor detection antibody.

Figure 59 shows IL- 15 -dependent proliferation of 32ϋb cells mediated by the 2ltl5-TGFRs complex (open squares) compared to IL-15 (black squares).

Figure 60 shows biological activity of the TGFpRII domain within the 2ltl 5-

TGFRs complex (open squares). TGFpRII/Fc (black squares) served as a positive control.

Figure 61A shows a flow cytometry graph of cell-surface CD25 expression of NK cells induced by the 2ltl5-TGFRs complex. Figure 61B shows a flow cytometry graph of cell-surface CD69 expression of NK cells induced by the 2ltl5-TGFRs complex.

Figure 62 shows a flow cytometry graph of intracellular interferon gamma expression of NK cells induced by the 2ltl5-TGFRs complex.

Figure 63 shows a bar graph showing cytotoxicity of 2ltl5-TGFRs-induced human NK cells against K562. K562 cells (labeled with Celltrace violet) at 1 x l0 ^A 5/well and human NK cells (NK cells were isolated from human blood huffy coat with StemCell human NK cell purification kit) at 2 x l0 ^A 5/well were incubated with l8tl5-l2s in RPMI-10 for 20 hours. Cell mixtures were harvested, stained with propidium iodide (PI), and analyzed using a BD FACSCelesta™ flow cytometer. The graph represents duplicated samples.

Figure 64 are schematic diagrams of an exemplary aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide. Figure 65 is a chromatograph showing the elution of an exemplary aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide from an anti -tissue factor affinity column.

Figure 66 is a chromatograph showing the elution of a Superdex 200 Increase 10/300 GL gel filtration column loaded with an exemplary aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide.

Figure 67 is a sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis- Tris gel) of an exemplary aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide purified using an anti-tissue factor affinity column.

Figure 68 is a graph showing the ELISA quantitation of an exemplary aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide performed using the methods described in Example 53. Purified tissue factor was used as the control.

Figure 69 is a graph showing the ability of an exemplary

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide to stimulate CD25 expression in CD4 ⁺ T-cells isolated from blood from two donors. The experiments were performed as described in Example 54.

Figure 70 is a graph showing the ability of an exemplary

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide to stimulate CD25 expression in CD8 ⁺ T-cells isolated from blood from two donors. The experiments were performed as described in Example 54.

Figure 71 is a graph showing the ability of an exemplary

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide to stimulate CD69 expression in CD4 ⁺ T-cells isolated from blood from two donors. The experiments were performed as described in Example 54.

Figure 72 shows a schematic diagram of an exemplary IL-7/IL-l5RaSu DNA construct.

Figure 73 shows a schematic diagram of an exemplary IL-21/TF/IL-15 DNA construct.

Figure 74 shows a schematic diagram of the interaction between the exemplary IL-7/IL-l5RaSu and IL-21/TF/IL-15 DNA constructs. Figure 75 shows a schematic diagram of the interaction between the exemplary IL-7/IL-l5RaSu and IL-21/TF/IL-15 fusion proteins resulting in an IL-2l/TF/IL-l5:IL- 7/IL-l5RaSu complex (2ltl5-7s).

Figure 76 shows a schematic diagram of an exemplary TGF-PRII/IL- 15RaSu DNA construct.

Figure 77 shows a schematic diagram of an exemplary IL-21/TF/IL-15 DNA construct.

Figure 78 shows a schematic diagram of the interaction between the exemplary TGF-b RII/IL-l5RaSu and IL-21/TF/IL-15 DNA constructs.

Figure 79 shows a schematic diagram of the interaction between the exemplary TGF-PRII /IL-l5RaSu and IL-21/TF/IL-15 fusion proteins resulting in an IL-21/TF/IL- 15: TGF-pRII/IL-l5RaSU complex (2ltl5-TGFRs).

Figure 80 shows a schematic diagram of an exemplary IL-2l/IL-l5RaSu DNA construct.

Figure 81 shows a schematic diagram of an exemplary IL-7/TF/IL-15 DNA construct.

Figure 82 shows a schematic diagram of the interaction between the exemplary IL-2l/IL-l5RaSu and IL-7/TF/IL-15 DNA constructs.

Figure 83 shows a schematic diagram of the interaction between the exemplary

IL-2l/IL-l5RaSu and IL-7/TF/IL-15 fusion proteins resulting in an IL-7/TF/IL-l5:IL- 2l/IL-l5RaSU complex (7tl5-2ls).

Figure 84 shows size exclusion chromatography (SEC) profiles of anti-TF IgGl antibody, 7tl 5-21 s and the complex containing equal amounts of anti-TF IgGl antibody and 7tl5-2ls.

Figure 85 shows a schematic of the 7tl5-l6s2l construct.

Figure 86 shows an additional schematic of the 7tl5-l6s2l construct.

Figures 87A and 87B are graphs showing binding of 7tl5-l6s2l to CHO cells expressing human CDl6b as compared to a control protein.

Figures 88A-88C are results from ELISA experiments using antibodies against

IL-15, IL-21, and IL-7 in detecting 7tl 5- 16s21. Figure 89 shows results of the 32 ϋb cell proliferation assay with 7tl5-l6s2l or recombinant IL- 15.

Figure 90 is a line graph showing the chromatographic profile of 7tl5-l6s2l protein containing cell culture supernatant following binding and elution on anti-TF antibody resin.

Figure 91 shows the analytical SEC Profile of 7tl5-l6s2l.

Figure 92 shows a schematic of the TGFRtl5-l6s2l construct.

Figure 93 shows an additional schematic of the TGFRtl5-l6s2l construct.

Figures 94A and 94B show binding affinity of TGFRtl5-l6s2l and 7tl 5-21 s with CHO cells expressing human CDl6b. Figure 94A shows binding affinity of TGFRtl5- l6s2l with CHO cells expressing human CDl6b. Figure 94B shows binding affinity of 7tl 5-21 s with CHO cells expressing human CDl6b.

Figure 95 shows results of TGF l inhibition by TGFRtl5-l6s2l and TGFR-Fc.

Figure 96 shows results of 32ϋb cell proliferation assay with TGFRtl5-l6s2l or recombinant IL- 15.

Figures 97A-97C show results of detecting IL-15, IL-21, and TGF RII in TGFRtl5-l6s2l with corresponding antibodies using ELISA.

Figure 98 is a line graph showing the chromatographic profile of TGFRtl5-l6s2l protein containing cell culture supernatant following binding and elution on anti-TF antibody resin.

Figure 99 shows results of a reduced SDS-PAGE analysis of TGFRtl5-l6s2l.

Figure 100 shows a schematic of the 7tl5-7s construct.

Figure 101 shows an additional schematic of the 7tl5-7s construct.

Figure 102 is a line graph showing the chromatographic profile of 7tl5-7s protein containing cell culture supernatant following binding and elution on anti-TF antibody resin.

Figure 103 shows detection of TF, IL-15 and IL-7 in 7tl5-7s using ELISA.

Figure 104A and 104B show spleen weight and the percentages of immune cell types in 7tl5-7s-treated and control-treated mice. Figure 115A shows spleen weight in mice treated with 7tl5-7s as compared to PBS control. Figure 115B shows the percentage of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells in mice treated with 7tl5-7s as compared to PBS control.

Figure 105 shows a schematic of the TGFRtl5-TGFRs construct.

Figure 106 shows an additional schematic of the TGFRtl5-TGFRs construct. Figure 107 shows results of TGF l inhibition by TGFRtl5-TGFRs and TGFR-

Fc.

Figure 108 shows results of 32ϋb cell proliferation assay with TGFRtl5-TGFRs or recombinant IL-15

Figures 109A and 109B show results of detecting IL-15 and TGF RII in

TGFRtl 5-TGFRs with corresponding antibodies using ELISA.

Figure 110 is a line graph showing the chromatographic profile of TGFRtl 5- TGFRs protein containing cell culture supernatant following binding and elution on anti- TF antibody resin.

Figure 111 shows the analytical SEC profile of TGFRtl 5-TGFRs.

Figure 112 shows TGFRtl 5-TGFRs before and after deglycosylation as analyzed by reduced SDS-PAGE.

Figures 113 A and 113B show spleen weight and the percentages of immune cell types in TGFRtl 5-TGFRs-treated and control -treated mice. Figure 113 A shows spleen weight in mice treated with TGFRtl5-TGFRs as compared to PBS control. Figure 113B shows the percentage of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells in mice treated with TGFRtl 5-TGFRs as compared to PBS control.

Figure 114A and 114B show the spleen weight and immunostimulation over 92 hours in mice treated with TGFRtl 5-TGFRs. Figure 114A shows spleen weight of mice treated with TGFRtl 5-TGFRs at 16, 24, 48, 72, and 92 hours after treatment. Figure 114B shows the percentages of immune cells in mice treated with TGFRtl 5-TGFRs at

16, 24, 48, 72, and 92 hours after treatment.

Figure 115A and 115B show Ki67 and Granzyme B expression in mice treated with TGFRtl 5-TGFRs over time.

Figure 116 shows enhancement of cytotoxicity of splenocytes by TGFRtl 5- TGFRs in C57BL/6 Mice. Figure 117 shows changes in tumor size in response to PBS treatment, chemotherapy alone, TGFRtl5-TGFRs alone, or chemotherapy and TGFRtl5-TGFRs combination, in a pancreatic cancer mouse model.

Figure 118 shows the cytotoxicity of NK cells isolated from mice treated with T GFRt 15 -TGFRs .

Figures 119A-119E show results of the in vivo efficacy of TGFRtl5-TGFRs in a melanoma mouse model. Figure 119A shows a schematic of the treatment regimen.

Figure 119B shows tumor volume over time following saline treatment, chemotherapy (DTX), or chemotherapy (DTX), TGFRtl5-TGFRs and TA99 (anti-TRPl antibody) combination treatment. Figure 119C shows peripheral blood analysis using fluorescently labeled antibodies against NK1.1. Figure 119D shows peripheral blood analysis using fluorescently labeled antibodies against CD8. Figure 119E shows peripheral blood analysis using fluorescently labeled antibodies against CD4.

Figure 120 shows a schematic of the 7tl5-2lsl37L (long version) construct. Figure 121 shows an additional schematic of the 7tl5-2lsl37L (long version) construct.

Figure 122 is a line graph showing the chromatographic profile of 7tl5-2lsl37L (long version) protein containing cell culture supernatant following binding and elution on anti-TF antibody resin.

Figure 123 shows the analytical SEC profile of 7tl5-2lsl37L (long version).

Figure 124 shows binding of 7tl5-2lsl37L (short version) to CD137L (4.1BBL)

Figures 125A-125C show detection of IL15, IL21, and IL7 in 7tl5-2lsl37L (short version) with ELISA. Figure 125A shows detection of IL15 in 7tl5-2lsl37L (short version) with ELISA. Figure 125B shows detection of IL21 in 7tl5-2lsl37L (short version) with ELISA. Figure 125C shows detection of IL7 in 7tl5-2lsl37L (short version) with ELISA.

Figure 126 shows results from a CTLL-2 cell proliferation assay.

Figure 127 shows the activity of 7tl5-lsl37L (short version) in promoting IL21R containing B9 cell proliferation.

Figure 128 shows a schematic of the 7tl5-TGFRs construct.

Figure 129 shows an additional schematic of the 7tl5-TGFRs construct. Figure 130 shows results of TGF l inhibition by 7tl5-TGFRs and TGFR-Fc. Figures 131A-131C show detection of IL-15, TGF RII, and IL-7 in 7tl5-TGFRs with ELISA.

Figure 132 shows results of 32ϋb cell proliferation assay with 7tl5-TGFRs or recombinant IL- 15.

Figure 133 is a line graph showing the chromatographic profile of 7tl5-TGFRs protein containing cell culture supernatant following binding and elution on anti-TF antibody resin.

Figure 134 shows 7tl5-TGFRs before and after deglycosylation as analyzed using reduced SDS-PAGE.

Figure 135 shows ELISA detection of IL-7, IL-15 and TGF RII in the 7tl5-TGFRs protein.

Figures 136A and 136B show spleen weight and the percentages of immune cell types in 7tl5-TGFRs-treated and control-treated mice. Figure 136A shows spleen weight in mice treated with 7tl5-TGFRs at various dosages, as compared to PBS control. Figure 136B shows the percentage of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells in mice treated with 7tl5-TGFRs at various dosages, as compared to PBS control.

Figures l37A and 137B show upregulation of CD44 expression of CD4 ⁺ and

CD8 ⁺ T cells by 7tl5-TGFRs in C57BL/6 mice.

Figures 138A and 138B show upregulation of Ki67 expression and Granzyme B expression of CD8 ⁺ T cells and NK Cells by 7tl5-TGFRs in C57BL/6 mice.

Figure 139 shows enhancement of cytotoxicity of splenocytes by 7tl5-TGFRs in C57BL/6 mice.

Figure 140 shows a schematic of the TGFRtl5-2lsl37L construct.

Figure 141 shows an additional schematic of the TGFRtl5-2lsl37L construct.

Figure 142 is a line graph showing the chromatographic profile of

TGFRtl5-2lsl37L protein containing cell culture supernatant following binding and elution on anti-TF antibody resin.

Figure 143 shows a schematic of the TGFRtl5-TGFRs2l construct. Figure 144 shows an additional schematic of the TGFRtl5-TGFRs2l

construct.

Figure 145 is a line graph showing the chromatographic profile of TGFRtl5- TGFRs2l protein containing cell culture supernatant following binding and elution on anti-TF antibody resin.

Figure 146 shows TGFRtl5-TGFRs2l before and after deglycosylation as analyzed by reduced SDS-PAGE.

Figures 147A and 147B show detection of components of TGFRtl5-TGFRs2l using ELISA.

Figures 148A and 148B show the percentages and proliferation of CD4 ⁺ T cells,

CD8 ⁺ T cells, and natural killer (NK) cells present in the spleen of control-treated and TGFRtl5-TGFRs2l -treated mice.

Figure 149 shows upregulation of Granzyme B expression of splenocytes in mice treated with TGFRtl5-TGFRs2l.

Figure 150 shows enhancement of cytotoxicity of splenocytes by TGFRtl5-

TGFRs2l in C57BL/6 Mice.

Figure 151 shows a schematic of the TGFRtl5-TGFRsl6 construct.

Figure 152 shows an additional schematic of the TGFRtl5-TGFRsl6 construct.

Figure 153 shows a schematic of the TGFRtl5-TGFRsl37L construct.

Figure 154 shows an additional schematic of the TGFRtl5-TGFRsl37L construct.

Figure 155 are schematic diagrams of an exemplary 2t2 single-chain chimeric polypeptide.

Figure 156 shows IL-2 activity in 2t2 as compared to recombinant IL-2 using a 32ϋb cell proliferation assay.

Figure 157 shows IL-2 activity in 2t2 as compared to recombinant IL-2 using a

CTLL-2 cell proliferation assay.

Figure 158 shows the fasting blood glucose levels in ApoE ^/_ mice fed with standard chow or a high fat diet and treated with a PBS control (untreated) or with 2t2.

Figure 159 shows the ratio of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T regulatory cells in blood lymphocytes from ApoE ^/_ mice fed with standard chow or a high fat diet and treated with a PBS control (untreated) or with 2t2. Figure 160 is a line graph showing the chromatographic profile of 2t2 protein containing cell culture supernatant following binding and elution on anti-TF antibody resin.

Figure 161 shows an analytical SEC profile of 2t2.

Figures 162A and 162B show reduced SDS-PAGE analysis of 2t2 before and after deglycosylation. Figure 162A shows reduced SDS-PAGE analysis of 2t2before deglycosylation. Figure 162B shows reduced SDS-PAGE analysis of 2t2 after deglycosylation.

Figures 163 A and 163B show results of immunostimulation in C57BL/6 mice using 2t2. Figure 163 A shows spleen weight following treatment with 2t2. Figure 163B shows the percentages of immune cell types following 2t2 treatment.

Figure 164 shows upregulation of CD25 expression of CD4 ⁺ T cells in mice treated with 2t2.

Figure 165 shows the pharmacokinetics of 2t2 in C57BL/6 mice.

Figures 166A and 166B show effects of 2t2 in attenuating the formation of high fat-induced atherosclerotic plaques in ApoE ^/_ mice. Figure 166A shows a representative view of atherosclerotic plaques from ApoE ^/_ mice fed with standard chow or a high fat diet and treated with either PBS control or 2t2. Figure 166B shows the results of quantitative analysis of atherosclerotic plaques of each group.

Figure 167 shows fasting glucose levels in 2t2 treated-mice as compared to control -treated mice.

Figure 168 shows the percentage of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs in blood lymphocytes from mice treated with 2t2 and control-treated mice.

Figure 169 are schematic diagrams of an exemplary 15tl 5 single-chain chimeric polypeptide.

Figure 170 shows the IL-15 activity of 15tl 5 as compared to recombinant IL-15 in a 32ϋb cell proliferation assay.

Figure 171 is a line graph showing the chromatographic profile of 15tl 5 protein containing cell culture supernatant following binding and elution on anti-TF resin.

Figures 172A and 172B show reduced SDS-PAGE analysis of 15tl 5 before and after deglycosylation. Figure 172A shows reduced SDS-PAGE analysis of 15tl 5 before deglycosylation. Figure 172B shows reduced SDS-PAGE analysis of 15tl 5 after deglycosylation.

Figure 173 is a set of histograms and a set of graphs showing the change in the surface phenotype of NK cells after stimulation with 18tl 5-l2s, l8tl5-l2sl6, and 7tl 5- 21 s + anti-TF antibody.

Figure 174 is a set of graphs showing changes in the surface phenotype of lymphocyte populations after stimulation with l8tl5-l2s; l8tl5-l2sl6; a mixture of single cytokines rhILl5, rhåLl8, and rhIL-l2; 7tl 5-21 s + anti-TF antibody; 7tl 5-21 s; or anti-TF antibody.

Figure 175 is a graph of the increase in proliferation of NK cells in vitro after stimulation with a multi-chain construct alone or in the presence of anti-TF IgGl or anti- TF IgG4.

Figure 176 is a graph of the increase in proliferation over time of NK cells in vitro after stimulation with the combination of 7tl 5-21 s and anti-TF antibody.

Figure 177 is a graph of the changes in surface phenotype on 7tl 5-21 s + anti-TF antibody-expanded NK cells.

Figure 178 is a schematic and a set of graphs showing the persistence of 7tl 5-21 s and anti-TF antibody-expanded NK cells in NSG mice following treatment with 7tl 5-21, T GFRt 15 -TGFRs or 2t2.

Figure 179 A and 179B is a set of images and a set of graphs showing killing of chemically-induced senescent human fibroblasts by 7tl 5-21 s + anti-TF antibody- expanded NK cells.

Figure 180A-180C is a set of images and a set of graphs showing killing of UV- induced senescent human fibroblast by 7tl 5-21 s + anti-TF antibody-expanded NK cells.

Figure 181 shows proliferation of NK cells upon stimulation with 7tl5-2ls, 7tl 5-

2ls + anti-TF antibody (IgGl), or 7tl 5-21 s + anti-TF antibody (IgG4).

Figure 182 shows NK cell expansion induced by the 7tl5-2ls + anti-TF antibody (IgGl) and by the 7tl5-2lsl37L (short version) + anti-TF antibody (IgGl).

Figure 183 shows clearance of Daudi tumor cells in NSG mice upon treatment with NK cells activated by the 7tl5-2ls and anti-TF antibody (IgGl) complex. Figures 184A-184E show improvement of skin texture in ApoE ^/_ mice fed with a Western diet and treated with 2t2 or TGFRtl5-TGFRs. Figure 184A is a representative picture of a chow diet-fed ApoE ^/_ mouse taken one week after PBS treatment. Figure 184B is a representative picture of a Western diet-fed ApoE ^/_ mouse taken one week after PBS treatment. Figure 184C shows a representative picture of a Western diet-fed ApoE ^/_ mouse taken one week after TGFRtl5-TGFRs treatment. Figure 184D shows a representative picture of a Western diet-fed ApoE ^/_ mouse taken one week after 2t2 treatment. Figure 184E shows a representative picture of a Western diet-fed ApoE ^/_ mouse taken one week after treatment with 2ltl5-TGFRs.

Figure 185 shows a graph of Factor X (FX) activation following treatment with single-chain or multi-chain chimeric polypeptides.

Figure 186 shows clotting time for a buffer with varying concentrations of Innovin in a prothrombin time (PT) test.

Figure 187 shows clotting time for multi-chain chimeric polypeptides in a PT Assay.

Figure 188 shows clotting time of the multi-chain chimeric polypeptides in a PT assay when mixed with 32DB cells.

Figure 189 shows clotting time of multi-chain chimeric polypeptides in a PT assay when mixed with human PBMC.

Figure 190 shows binding of 7tl5-2lsl37L (long version) and 7tl5-2lsl37L

(short version) to CD137 (4.1BB).

Figure 191A-191D show detection of IL7, IL21, IL15, and 4.1BBL in 7tl5- 2lsl37L (long version) by the respective antibodies using ELISA.

Figure 192 shows IL-15 activity of 7tl5-2lsl37L (long version) and 7tl 5- 2lsl37L (short version) as evaluated by a IL2Ra y-containing CTLL2 cell proliferation assay.

Figure 193A-193C show human blood lymphocyte pStat5a responses in

CD4 ⁺ CD25 ^hi Treg cells, CD4 ⁺ CD25 T _COn cells, or in CD8 ⁺ T _COn cells in response to 2t2 or IL2 treatment. Figure 193 A shows pSTAT5 responses in CD4 ⁺ CD25 ^hi T _reg cells. Figure 193B shows pSTAT5 responses in CD4 ⁺ CD25 T _COn cells. Figure 193C shows pSTAT5 responses in CD8 ⁺ T _COn cells. Figure 194 is a graph showing the percentage different in DNA demethylation in NK cells (relative to unexposed NK cells) from two different donors following expansion with 7tl 5-21 s+ anti-tissue factor (TF)-antibody (IgGl) (50 nM).

DETAILED DESCRIPTION

Provided herein are a variety of single-chain and multi-chain chimeric

polypeptides, methods of using them, and kits including them. In some embodiments, the single-chain and multi-chain chimeric polypeptides include a linker domain positioned between two target-binding domains. In some embodiments, the linker domain is or includes a soluble tissue factor domain. In some embodiments, the linker domain can be recognized by a cognate IgGl antibody (e.g., a monoclonal antibody). In some embodiments, a single-chain or multi-chain chimeric polypeptide provided herein can be used to stimulate an immune cell, induce or increase proliferation of an immune cell, induce differentiation of an immune cell, or treat a subject in need thereof (e.g., a subject having cancer or an aging-related disease or condition). Single Chain Chimeric Polypeptides

Some embodiments of any of the methods described herein include the use of any of the single chain chimeric polypeptides described herein. Some embodiments of any of the kits described herein include any of the single-chain chimeric polypeptides described herein. The single-chain chimeric polypeptides provided herein include a first target- binding domain, a linker domain, and a second target-binding domain, where the first target-binding domain and the second target-binding domain are each independently selected from the group consisting of a soluble interleukin or cytokine protein, an antigen-binding domain, and a soluble interleukin or cytokine receptor, and ligands of co stimulatory molecules.

In some examples of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide can have a total length of about 50 amino acids to about 3000 amino acids, about 50 amino acids to about 2500 amino acids, about 50 amino acids to about 2000 amino acids, about 50 amino acids to about 1500 amino acids, about 50 amino acids to about 1000 amino acids, about 50 amino acids to about 950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to about 850 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 480 amino acids, about 50 amino acids to about 460 amino acids, about 50 amino acids to about 440 amino acids, about 50 amino acids to about 420 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 380 amino acids, about 50 amino acids to about 360 amino acids, about 50 amino acids to about 340 amino acids, about 50 amino acids to about 320 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 3000 amino acids, about 100 amino acids to about 2500 amino acids, about 100 amino acids to about 2000 amino acids, about 100 amino acids to about 1500 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 480 amino acids, about 100 amino acids to about 460 amino acids, about 100 amino acids to about 440 amino acids, about 100 amino acids to about 420 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 380 amino acids, about 100 amino acids to about 360 amino acids, about 100 amino acids to about 340 amino acids, about 100 amino acids to about 320 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 280 amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 3000 amino acids, about 150 amino acids to about 2500 amino acids, about 150 amino acids to about 2000 amino acids, about 150 amino acids to about 1500 amino acids, about 150 amino acids to about 1000 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids to about 850 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 480 amino acids, about 150 amino acids to about 460 amino acids, about 150 amino acids to about 440 amino acids, about 150 amino acids to about 420 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 380 amino acids, about 150 amino acids to about 360 amino acids, about 150 amino acids to about 340 amino acids, about 150 amino acids to about 320 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 280 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 200 amino acids to about 3000 amino acids, about 200 amino acids to about 2500 amino acids, about 200 amino acids to about 2000 amino acids, about 200 amino acids to about 1500 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 480 amino acids, about 200 amino acids to about 460 amino acids, about 200 amino acids to about 440 amino acids, about 200 amino acids to about 420 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 380 amino acids, about 200 amino acids to about 360 amino acids, about 200 amino acids to about 340 amino acids, about 200 amino acids to about 320 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 3000 amino acids, about 220 amino acids to about 2500 amino acids, about 220 amino acids to about 2000 amino acids, about 220 amino acids to about 1500 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 amino acids, about 220 amino acids to about 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 500 amino acids, about 220 amino acids to about 480 amino acids, about 220 amino acids to about 460 amino acids, about 220 amino acids to about 440 amino acids, about 220 amino acids to about 420 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 380 amino acids, about 220 amino acids to about 360 amino acids, about 220 amino acids to about 340 amino acids, about 220 amino acids to about 320 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 3000 amino acids, about 240 amino acids to about 2500 amino acids, about 240 amino acids to about 2000 amino acids, about 240 amino acids to about 1500 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids, about 240 amino acids to about 480 amino acids, about 240 amino acids to about 460 amino acids, about 240 amino acids to about 440 amino acids, about 240 amino acids to about 420 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 380 amino acids, about 240 amino acids to about 360 amino acids, about 240 amino acids to about 340 amino acids, about 240 amino acids to about 320 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 3000 amino acids, about 260 amino acids to about 2500 amino acids, about 260 amino acids to about 2000 amino acids, about 260 amino acids to about 1500 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 480 amino acids, about 260 amino acids to about 460 amino acids, about 260 amino acids to about 440 amino acids, about 260 amino acids to about 420 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 380 amino acids, about 260 amino acids to about 360 amino acids, about 260 amino acids to about 340 amino acids, about 260 amino acids to about 320 amino acids, about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 3000 amino acids, about 280 amino acids to about 2500 amino acids, about 280 amino acids to about 2000 amino acids, about 280 amino acids to about 1500 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 480 amino acids, about 280 amino acids to about 460 amino acids, about 280 amino acids to about 440 amino acids, about 280 amino acids to about 420 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 380 amino acids, about 280 amino acids to about 360 amino acids, about 280 amino acids to about 340 amino acids, about 280 amino acids to about 320 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 3000 amino acids, about 300 amino acids to about 2500 amino acids, about 300 amino acids to about 2000 amino acids, about 300 amino acids to about 1500 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 480 amino acids, about 300 amino acids to about 460 amino acids, about 300 amino acids to about 440 amino acids, about 300 amino acids to about 420 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 380 amino acids, about 300 amino acids to about 360 amino acids, about 300 amino acids to about 340 amino acids, about 300 amino acids to about 320 amino acids, about 320 amino acids to about 3000 amino acids, about 320 amino acids to about 2500 amino acids, about 320 amino acids to about 2000 amino acids, about 320 amino acids to about 1500 amino acids, about 320 amino acids to about 1000 amino acids, about 320 amino acids to about 950 amino acids, about 320 amino acids to about 900 amino acids, about 320 amino acids to about 850 amino acids, about 320 amino acids to about 800 amino acids, about 320 amino acids to about 750 amino acids, about 320 amino acids to about 700 amino acids, about 320 amino acids to about 650 amino acids, about 320 amino acids to about 600 amino acids, about 320 amino acids to about 550 amino acids, about 320 amino acids to about 500 amino acids, about 320 amino acids to about 480 amino acids, about 320 amino acids to about 460 amino acids, about 320 amino acids to about 440 amino acids, about 320 amino acids to about 420 amino acids, about 320 amino acids to about 400 amino acids, about 320 amino acids to about 380 amino acids, about 320 amino acids to about 360 amino acids, about 320 amino acids to about 340 amino acids, about 340 amino acids to about 3000 amino acids, about 340 amino acids to about 2500 amino acids, about 340 amino acids to about 2000 amino acids, about 340 amino acids to about 1500 amino acids, about 340 amino acids to about 1000 amino acids, about 340 amino acids to about 950 amino acids, about 340 amino acids to about 900 amino acids, about 340 amino acids to about 850 amino acids, about 340 amino acids to about 800 amino acids, about 340 amino acids to about 750 amino acids, about 340 amino acids to about 700 amino acids, about 340 amino acids to about 650 amino acids, about 340 amino acids to about 600 amino acids, about 340 amino acids to about 550 amino acids, about 340 amino acids to about 500 amino acids, about 340 amino acids to about 480 amino acids, about 340 amino acids to about 460 amino acids, about 340 amino acids to about 440 amino acids, about 340 amino acids to about 420 amino acids, about 340 amino acids to about 400 amino acids, about 340 amino acids to about 380 amino acids, about 340 amino acids to about 360 amino acids, about 360 amino acids to about 3000 amino acids, about 360 amino acids to about 2500 amino acids, about 360 amino acids to about 2000 amino acids, about 360 amino acids to about 1500 amino acids, about 360 amino acids to about 1000 amino acids, about 360 amino acids to about 950 amino acids, about 360 amino acids to about 900 amino acids, about 360 amino acids to about 850 amino acids, about 360 amino acids to about 800 amino acids, about 360 amino acids to about 750 amino acids, about 360 amino acids to about 700 amino acids, about 360 amino acids to about 650 amino acids, about 360 amino acids to about 600 amino acids, about 360 amino acids to about 550 amino acids, about 360 amino acids to about 500 amino acids, about 360 amino acids to about 480 amino acids, about 360 amino acids to about 460 amino acids, about 360 amino acids to about 440 amino acids, about 360 amino acids to about 420 amino acids, about 360 amino acids to about 400 amino acids, about 360 amino acids to about 380 amino acids, about 380 amino acids to about 3000 amino acids, about 380 amino acids to about 2500 amino acids, about 380 amino acids to about 2000 amino acids, about 380 amino acids to about 1500 amino acids, about 380 amino acids to about 1000 amino acids, about 380 amino acids to about 950 amino acids, about 380 amino acids to about 900 amino acids, about 380 amino acids to about 850 amino acids, about 380 amino acids to about 800 amino acids, about 380 amino acids to about 750 amino acids, about 380 amino acids to about 700 amino acids, about 380 amino acids to about 650 amino acids, about 380 amino acids to about 600 amino acids, about 380 amino acids to about 550 amino acids, about 380 amino acids to about 500 amino acids, about 380 amino acids to about 480 amino acids, about 380 amino acids to about 460 amino acids, about 380 amino acids to about 440 amino acids, about 380 amino acids to about 420 amino acids, about 380 amino acids to about 400 amino acids, about 400 amino acids to about 3000 amino acids, about 400 amino acids to about 2500 amino acids, about 400 amino acids to about 2000 amino acids, about 400 amino acids to about 1500 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 480 amino acids, about 400 amino acids to about 460 amino acids, about 400 amino acids to about 440 amino acids, about 400 amino acids to about 420 amino acids, about 420 amino acids to about 3000 amino acids, about 420 amino acids to about 2500 amino acids, about 420 amino acids to about 2000 amino acids, about 420 amino acids to about 1500 amino acids, about 420 amino acids to about 1000 amino acids, about 420 amino acids to about 950 amino acids, about 420 amino acids to about 900 amino acids, about 420 amino acids to about 850 amino acids, about 420 amino acids to about 800 amino acids, about 420 amino acids to about 750 amino acids, about 420 amino acids to about 700 amino acids, about 420 amino acids to about 650 amino acids, about 420 amino acids to about 600 amino acids, about 420 amino acids to about 550 amino acids, about 420 amino acids to about 500 amino acids, about 420 amino acids to about 480 amino acids, about 420 amino acids to about 460 amino acids, about 420 amino acids to about 440 amino acids, about 440 amino acids to about 3000 amino acids, about 440 amino acids to about 2500 amino acids, about 440 amino acids to about 2000 amino acids, about 440 amino acids to about 1500 amino acids, about 440 amino acids to about 1000 amino acids, about 440 amino acids to about 950 amino acids, about 440 amino acids to about 900 amino acids, about 440 amino acids to about 850 amino acids, about 440 amino acids to about 800 amino acids, about 440 amino acids to about 750 amino acids, about 440 amino acids to about 700 amino acids, about 440 amino acids to about 650 amino acids, about 440 amino acids to about 600 amino acids, about 440 amino acids to about 550 amino acids, about 440 amino acids to about 500 amino acids, about 440 amino acids to about 480 amino acids, about 440 amino acids to about 460 amino acids, about 460 amino acids to about 3000 amino acids, about 460 amino acids to about 2500 amino acids, about 460 amino acids to about 2000 amino acids, about 460 amino acids to about 1500 amino acids, about 460 amino acids to about 1000 amino acids, about 460 amino acids to about 950 amino acids, about 460 amino acids to about 900 amino acids, about 460 amino acids to about 850 amino acids, about 460 amino acids to about 800 amino acids, about 460 amino acids to about 750 amino acids, about 460 amino acids to about 700 amino acids, about 460 amino acids to about 650 amino acids, about 460 amino acids to about 600 amino acids, about 460 amino acids to about 550 amino acids, about 460 amino acids to about 500 amino acids, about 460 amino acids to about 480 amino acids, about 480 amino acids to about 3000 amino acids, about 480 amino acids to about 2500 amino acids, about 480 amino acids to about 2000 amino acids, about 480 amino acids to about 1500 amino acids, about 480 amino acids to about 1000 amino acids, about 480 amino acids to about 950 amino acids, about 480 amino acids to about 900 amino acids, about 480 amino acids to about 850 amino acids, about 480 amino acids to about 800 amino acids, about 480 amino acids to about 750 amino acids, about 480 amino acids to about 700 amino acids, about 480 amino acids to about 650 amino acids, about 480 amino acids to about 600 amino acids, about 480 amino acids to about 550 amino acids, about 480 amino acids to about 500 amino acids, about 500 amino acids to about 3000 amino acids, about 500 amino acids to about 2500 amino acids, about 500 amino acids to about 2000 amino acids, about 500 amino acids to about 1500 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to about 3000 amino acids, about 550 amino acids to about 2500 amino acids, about 550 amino acids to about 2000 amino acids, about 550 amino acids to about 1500 amino acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 3000 amino acids, about 600 amino acids to about 2500 amino acids, about 600 amino acids to about 2000 amino acids, about 600 amino acids to about 1500 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 3000 amino acids, about 650 amino acids to about 2500 amino acids, about 650 amino acids to about 2000 amino acids, about 650 amino acids to about 1500 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 3000 amino acids, about 700 amino acids to about 2500 amino acids, about 700 amino acids to about 2000 amino acids, about 700 amino acids to about 1500 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 3000 amino acids, about 750 amino acids to about 2500 amino acids, about 750 amino acids to about 2000 amino acids, about 750 amino acids to about 1500 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids, about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 3000 amino acids, about 800 amino acids to about 2500 amino acids, about 800 amino acids to about 2000 amino acids, about 800 amino acids to about 1500 amino acids, about 800 amino acids to about 1000 amino acids, about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 3000 amino acids, about 850 amino acids to about 2500 amino acids, about 850 amino acids to about 2000 amino acids, about 850 amino acids to about 1500 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 3000 amino acids, about 900 amino acids to about 2500 amino acids, about 900 amino acids to about 2000 amino acids, about 900 amino acids to about 1500 amino acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids, about 950 amino acids to about 3000 amino acids, about 950 amino acids to about 2500 amino acids, about 950 amino acids to about 2000 amino acids, about 950 amino acids to about 1500 amino acids, about 950 amino acids to about 1000 amino acids, about 1000 amino acids to about 3000 amino acids, about 1000 amino acids to about 2500 amino acids, about 1000 amino acids to about 2000 amino acids, about 1000 amino acids to about 1500 amino acids, about 1500 amino acids to about 3000 amino acids, about 1500 amino acids to about 2500 amino acids, about 1500 amino acids to about 2000 amino acids, about 2000 amino acids to about 3000 amino acids, about 2000 amino acids to about 2500 amino acids, or about 2500 amino acids to about 3000 amino acids.

In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) and the linker domain (e.g., any of the exemplary linker described herein) directly abut each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) and the linker domain (e.g., any of the exemplary linker domains described herein). In some embodiments of any of the single-chain chimeric polypeptides described herein, the linker domain (e.g., any of the exemplary linker domains described herein) and the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) directly abut each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the linker domain (e.g., any of the exemplary linker domains described herein) and the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art).

In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) and the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) directly abut each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) and the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art). In some embodiments of any of the single-chain chimeric polypeptides described herein, the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) and the linker domain (e.g., any of the exemplary linker domains described herein) directly abut each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) and the linker domain (e.g., any of the exemplary linker domains described herein or known in the art).

Non-limiting aspects of these chimeric polypeptides, nucleic acids, vectors, cells, and methods are described below, and can be used in any combination without limitation. Additional aspects of these chimeric polypeptides, nucleic acids, vectors, cells, and methods are known in the art. Multi-Chain Chimeric Polypeptides

Some embodiments of any of the methods described herein include the use of any of the multi-chain chimeric polypeptides described herein. Some embodiments of any of the kits described herein include any of the multi-chain chimeric polypeptides described herein.

The multi-chain chimeric polypeptides provided herein include: (a) a first chimeric polypeptide including: (i) a first target-binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target- binding domain, where the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains.

In some examples of any of the multi-chain chimeric polypeptides described herein the total length of first chimeric polypeptide and/or the second chimeric polypeptide can each independently be about 50 amino acids to about 3000 amino acids, about 50 amino acids to about 2500 amino acids, about 50 amino acids to about 2000 amino acids, about 50 amino acids to about 1500 amino acids, about 50 amino acids to about 1000 amino acids, about 50 amino acids to about 950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to about 850 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 480 amino acids, about 50 amino acids to about 460 amino acids, about 50 amino acids to about 440 amino acids, about 50 amino acids to about 420 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 380 amino acids, about 50 amino acids to about 360 amino acids, about 50 amino acids to about 340 amino acids, about 50 amino acids to about 320 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 3000 amino acids, about 100 amino acids to about 2500 amino acids, about 100 amino acids to about 2000 amino acids, about 100 amino acids to about 1500 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 480 amino acids, about 100 amino acids to about 460 amino acids, about 100 amino acids to about 440 amino acids, about 100 amino acids to about 420 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 380 amino acids, about 100 amino acids to about 360 amino acids, about 100 amino acids to about 340 amino acids, about 100 amino acids to about 320 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 280 amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 3000 amino acids, about 150 amino acids to about 2500 amino acids, about 150 amino acids to about 2000 amino acids, about 150 amino acids to about 1500 amino acids, about 150 amino acids to about 1000 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids to about 850 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 480 amino acids, about 150 amino acids to about 460 amino acids, about 150 amino acids to about 440 amino acids, about 150 amino acids to about 420 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 380 amino acids, about 150 amino acids to about 360 amino acids, about 150 amino acids to about 340 amino acids, about 150 amino acids to about 320 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 280 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 200 amino acids to about 3000 amino acids, about 200 amino acids to about 2500 amino acids, about 200 amino acids to about 2000 amino acids, about 200 amino acids to about 1500 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 480 amino acids, about 200 amino acids to about 460 amino acids, about 200 amino acids to about 440 amino acids, about 200 amino acids to about 420 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 380 amino acids, about 200 amino acids to about 360 amino acids, about 200 amino acids to about 340 amino acids, about 200 amino acids to about 320 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 3000 amino acids, about 220 amino acids to about 2500 amino acids, about 220 amino acids to about 2000 amino acids, about 220 amino acids to about 1500 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 amino acids, about 220 amino acids to about 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 500 amino acids, about 220 amino acids to about 480 amino acids, about 220 amino acids to about 460 amino acids, about 220 amino acids to about 440 amino acids, about 220 amino acids to about 420 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 380 amino acids, about 220 amino acids to about 360 amino acids, about 220 amino acids to about 340 amino acids, about 220 amino acids to about 320 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 3000 amino acids, about 240 amino acids to about 2500 amino acids, about 240 amino acids to about 2000 amino acids, about 240 amino acids to about 1500 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids, about 240 amino acids to about 480 amino acids, about 240 amino acids to about 460 amino acids, about 240 amino acids to about 440 amino acids, about 240 amino acids to about 420 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 380 amino acids, about 240 amino acids to about 360 amino acids, about 240 amino acids to about 340 amino acids, about 240 amino acids to about 320 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 3000 amino acids, about 260 amino acids to about 2500 amino acids, about 260 amino acids to about 2000 amino acids, about 260 amino acids to about 1500 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 480 amino acids, about 260 amino acids to about 460 amino acids, about 260 amino acids to about 440 amino acids, about 260 amino acids to about 420 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 380 amino acids, about 260 amino acids to about 360 amino acids, about 260 amino acids to about 340 amino acids, about 260 amino acids to about 320 amino acids, about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 3000 amino acids, about 280 amino acids to about 2500 amino acids, about 280 amino acids to about 2000 amino acids, about 280 amino acids to about 1500 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 480 amino acids, about 280 amino acids to about 460 amino acids, about 280 amino acids to about 440 amino acids, about 280 amino acids to about 420 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 380 amino acids, about 280 amino acids to about 360 amino acids, about 280 amino acids to about 340 amino acids, about 280 amino acids to about 320 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 3000 amino acids, about 300 amino acids to about 2500 amino acids, about 300 amino acids to about 2000 amino acids, about 300 amino acids to about 1500 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 480 amino acids, about 300 amino acids to about 460 amino acids, about 300 amino acids to about 440 amino acids, about 300 amino acids to about 420 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 380 amino acids, about 300 amino acids to about 360 amino acids, about 300 amino acids to about 340 amino acids, about 300 amino acids to about 320 amino acids, about 320 amino acids to about 3000 amino acids, about 320 amino acids to about 2500 amino acids, about 320 amino acids to about 2000 amino acids, about 320 amino acids to about 1500 amino acids, about 320 amino acids to about 1000 amino acids, about 320 amino acids to about 950 amino acids, about 320 amino acids to about 900 amino acids, about 320 amino acids to about 850 amino acids, about 320 amino acids to about 800 amino acids, about 320 amino acids to about 750 amino acids, about 320 amino acids to about 700 amino acids, about 320 amino acids to about 650 amino acids, about 320 amino acids to about 600 amino acids, about 320 amino acids to about 550 amino acids, about 320 amino acids to about 500 amino acids, about 320 amino acids to about 480 amino acids, about 320 amino acids to about 460 amino acids, about 320 amino acids to about 440 amino acids, about 320 amino acids to about 420 amino acids, about 320 amino acids to about 400 amino acids, about 320 amino acids to about 380 amino acids, about 320 amino acids to about 360 amino acids, about 320 amino acids to about 340 amino acids, about 340 amino acids to about 3000 amino acids, about 340 amino acids to about 2500 amino acids, about 340 amino acids to about 2000 amino acids, about 340 amino acids to about 1500 amino acids, about 340 amino acids to about 1000 amino acids, about 340 amino acids to about 950 amino acids, about 340 amino acids to about 900 amino acids, about 340 amino acids to about 850 amino acids, about 340 amino acids to about 800 amino acids, about 340 amino acids to about 750 amino acids, about 340 amino acids to about 700 amino acids, about 340 amino acids to about 650 amino acids, about 340 amino acids to about 600 amino acids, about 340 amino acids to about 550 amino acids, about 340 amino acids to about 500 amino acids, about 340 amino acids to about 480 amino acids, about 340 amino acids to about 460 amino acids, about 340 amino acids to about 440 amino acids, about 340 amino acids to about 420 amino acids, about 340 amino acids to about 400 amino acids, about 340 amino acids to about 380 amino acids, about 340 amino acids to about 360 amino acids, about 360 amino acids to about 3000 amino acids, about 360 amino acids to about 2500 amino acids, about 360 amino acids to about 2000 amino acids, about 360 amino acids to about 1500 amino acids, about 360 amino acids to about 1000 amino acids, about 360 amino acids to about 950 amino acids, about 360 amino acids to about 900 amino acids, about 360 amino acids to about 850 amino acids, about 360 amino acids to about 800 amino acids, about 360 amino acids to about 750 amino acids, about 360 amino acids to about 700 amino acids, about 360 amino acids to about 650 amino acids, about 360 amino acids to about 600 amino acids, about 360 amino acids to about 550 amino acids, about 360 amino acids to about 500 amino acids, about 360 amino acids to about 480 amino acids, about 360 amino acids to about 460 amino acids, about 360 amino acids to about 440 amino acids, about 360 amino acids to about 420 amino acids, about 360 amino acids to about 400 amino acids, about 360 amino acids to about 380 amino acids, about 380 amino acids to about 3000 amino acids, about 380 amino acids to about 2500 amino acids, about 380 amino acids to about 2000 amino acids, about 380 amino acids to about 1500 amino acids, about 380 amino acids to about 1000 amino acids, about 380 amino acids to about 950 amino acids, about 380 amino acids to about 900 amino acids, about 380 amino acids to about 850 amino acids, about 380 amino acids to about 800 amino acids, about 380 amino acids to about 750 amino acids, about 380 amino acids to about 700 amino acids, about 380 amino acids to about 650 amino acids, about 380 amino acids to about 600 amino acids, about 380 amino acids to about 550 amino acids, about 380 amino acids to about 500 amino acids, about 380 amino acids to about 480 amino acids, about 380 amino acids to about 460 amino acids, about 380 amino acids to about 440 amino acids, about 380 amino acids to about 420 amino acids, about 380 amino acids to about 400 amino acids, about 400 amino acids to about 3000 amino acids, about 400 amino acids to about 2500 amino acids, about 400 amino acids to about 2000 amino acids, about 400 amino acids to about 1500 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 480 amino acids, about 400 amino acids to about 460 amino acids, about 400 amino acids to about 440 amino acids, about 400 amino acids to about 420 amino acids, about 420 amino acids to about 3000 amino acids, about 420 amino acids to about 2500 amino acids, about 420 amino acids to about 2000 amino acids, about 420 amino acids to about 1500 amino acids, about 420 amino acids to about 1000 amino acids, about 420 amino acids to about 950 amino acids, about 420 amino acids to about 900 amino acids, about 420 amino acids to about 850 amino acids, about 420 amino acids to about 800 amino acids, about 420 amino acids to about 750 amino acids, about 420 amino acids to about 700 amino acids, about 420 amino acids to about 650 amino acids, about 420 amino acids to about 600 amino acids, about 420 amino acids to about 550 amino acids, about 420 amino acids to about 500 amino acids, about 420 amino acids to about 480 amino acids, about 420 amino acids to about 460 amino acids, about 420 amino acids to about 440 amino acids, about 440 amino acids to about 3000 amino acids, about 440 amino acids to about 2500 amino acids, about 440 amino acids to about 2000 amino acids, about 440 amino acids to about 1500 amino acids, about 440 amino acids to about 1000 amino acids, about 440 amino acids to about 950 amino acids, about 440 amino acids to about 900 amino acids, about 440 amino acids to about 850 amino acids, about 440 amino acids to about 800 amino acids, about 440 amino acids to about 750 amino acids, about 440 amino acids to about 700 amino acids, about 440 amino acids to about 650 amino acids, about 440 amino acids to about 600 amino acids, about 440 amino acids to about 550 amino acids, about 440 amino acids to about 500 amino acids, about 440 amino acids to about 480 amino acids, about 440 amino acids to about 460 amino acids, about 460 amino acids to about 3000 amino acids, about 460 amino acids to about 2500 amino acids, about 460 amino acids to about 2000 amino acids, about 460 amino acids to about 1500 amino acids, about 460 amino acids to about 1000 amino acids, about 460 amino acids to about 950 amino acids, about 460 amino acids to about 900 amino acids, about 460 amino acids to about 850 amino acids, about 460 amino acids to about 800 amino acids, about 460 amino acids to about 750 amino acids, about 460 amino acids to about 700 amino acids, about 460 amino acids to about 650 amino acids, about 460 amino acids to about 600 amino acids, about 460 amino acids to about 550 amino acids, about 460 amino acids to about 500 amino acids, about 460 amino acids to about 480 amino acids, about 480 amino acids to about 3000 amino acids, about 480 amino acids to about 2500 amino acids, about 480 amino acids to about 2000 amino acids, about 480 amino acids to about 1500 amino acids, about 480 amino acids to about 1000 amino acids, about 480 amino acids to about 950 amino acids, about 480 amino acids to about 900 amino acids, about 480 amino acids to about 850 amino acids, about 480 amino acids to about 800 amino acids, about 480 amino acids to about 750 amino acids, about 480 amino acids to about 700 amino acids, about 480 amino acids to about 650 amino acids, about 480 amino acids to about 600 amino acids, about 480 amino acids to about 550 amino acids, about 480 amino acids to about 500 amino acids, about 500 amino acids to about 3000 amino acids, about 500 amino acids to about 2500 amino acids, about 500 amino acids to about 2000 amino acids, about 500 amino acids to about 1500 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to about 3000 amino acids, about 550 amino acids to about 2500 amino acids, about 550 amino acids to about 2000 amino acids, about 550 amino acids to about 1500 amino acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 3000 amino acids, about 600 amino acids to about 2500 amino acids, about 600 amino acids to about 2000 amino acids, about 600 amino acids to about 1500 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 3000 amino acids, about 650 amino acids to about 2500 amino acids, about 650 amino acids to about 2000 amino acids, about 650 amino acids to about 1500 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 3000 amino acids, about 700 amino acids to about 2500 amino acids, about 700 amino acids to about 2000 amino acids, about 700 amino acids to about 1500 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 3000 amino acids, about 750 amino acids to about 2500 amino acids, about 750 amino acids to about 2000 amino acids, about 750 amino acids to about 1500 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids, about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 3000 amino acids, about 800 amino acids to about 2500 amino acids, about 800 amino acids to about 2000 amino acids, about 800 amino acids to about 1500 amino acids, about 800 amino acids to about 1000 amino acids, about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 3000 amino acids, about 850 amino acids to about 2500 amino acids, about 850 amino acids to about 2000 amino acids, about 850 amino acids to about 1500 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 3000 amino acids, about 900 amino acids to about 2500 amino acids, about 900 amino acids to about 2000 amino acids, about 900 amino acids to about 1500 amino acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids, about 950 amino acids to about 3000 amino acids, about 950 amino acids to about 2500 amino acids, about 950 amino acids to about 2000 amino acids, about 950 amino acids to about 1500 amino acids, about 950 amino acids to about 1000 amino acids, about 1000 amino acids to about 3000 amino acids, about 1000 amino acids to about 2500 amino acids, about 1000 amino acids to about 2000 amino acids, about 1000 amino acids to about 1500 amino acids, about 1500 amino acids to about 3000 amino acids, about 1500 amino acids to about 2500 amino acids, about 1500 amino acids to about 2000 amino acids, about 2000 amino acids to about 3000 amino acids, about 2000 amino acids to about 2500 amino acids, or about 2500 amino acids to about 3000 amino acids. Diagrams of exemplary multi-chain chimeric polypeptides provided herein are depicted in Figures 1 and 2.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain (e.g., any of the first target-binding domains described herein) and the linker domain (e.g., any of the exemplary linker domains described herein) directly abut each other in the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target-binding domain (e.g., any of the exemplary first target-binding domains described herein) and the linker domain (e.g., any of the exemplary linker domains described herein) in the first chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the linker domain (e.g., any of the exemplary linker domains described herein) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) directly abut each other in the first chimeric polypeptide. In some embodiments of any of the multi- chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the linker domain (e.g., any of the exemplary linker domains described herein) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) and the second target-binding domain (e.g., any of the exemplary second target-binding domains described herein) directly abut each other in the second chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) and the second target- binding domain (e.g., any of the exemplary second target-binding domains described herein) in the second chimeric polypeptide.

Tissue Factor

Human tissue factor is a 263 amino-acid transmembrane protein containing three domains: (1) a 2l9-amino acid N-terminal extracellular domain (residues 1-219); (2) a 22-amino acid transmembrane domain (residues 220-242); and (3) a 21 -amino acid cytoplasmic C-terminal tail (residues 242-263) ((UniProtKB Identifier Number: P13726). The cytoplasmic tail contains two phosphorylation sites at Ser ²⁵³ and Ser ²⁵⁸ , and one S- palmitoylation site at Cys ²⁴⁵ . Deletion or mutation of the cytoplasmic domain was not found to affect tissue factor coagulation activity. Tissue factor has one S-palmitoylation site in the intracellular domain of the protein at Cys ²⁴⁵ . The Cys ²⁴⁵ is located at the amino acid terminus of the intracellular domain and close to the membrane surface. The tissue factor transmembrane domain is composed of a single-spanning a-helix.

The extracellular domain of tissue factor, composed of two fibronectin type III domains, is connected to the transmembrane domain through a six-amino acid linker.

This linker provides conformational flexibility to decouple the tissue factor extracellular domain from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module is composed of two overlapping b sheets with the top sheet domain containing three antiparallel b-strands and the bottom sheet containing four b-strands.

The b-strands are connected by b-loops between strand bA and bB, bE and bϋ, and bE and bE, all of which are conserved in conformation in the two modules. There are three short a-helix segments connecting the b-strands. A unique feature of tissue factor is a 17- amino acid b-hairpin between strand bΐq and strand bΐ 1, which is not a common element of the fibronectin superfamily. The N-terminal domain also contains a 12 amino acid loop between b6T and b70 that is not present in the C-terminal domain and is unique to tissue factor. Such a fibronectin type III domain structure is a feature of the

immunoglobulin-like family of protein folds and is conserved among a wide variety of extracellular proteins.

The zymogen FVII is rapidly converted to FVIIa by limited proteolysis once it binds to tissue to form the active tissue factor-FVIIa complex. The FVIIa, which circulates as an enzyme at a concentration of approximately 0.1 nM (1% of plasma FVII), can also bind directly to tissue factor. The allosteric interaction between tissue factor and FVIIa on the tissue factor-FVIIa complex greatly increases the enzymatic activity of FVIIa: an approximate 20- to lOO-fold increase in the rate of hydrolysis of small, chromogenic peptidyl substrates, and nearly a million-fold increase in the rate of activation of the natural macromolecular substrates FIX and FX. In concert with allosteric activation of the active site of FVIIa upon binding to tissue factor, the formation of tissue factor-FVIIa complex on phospholipid bilayer (i.e., upon exposure of phosphatidyl-L-serine on membrane surfaces) increases the rate of FIX or FX activation, in a Ca ²⁺ -dependent manner, an additional 1, 000-fold. The roughly million-fold overall increase in FX activation by tissue factor-FVIIa-phospholipid complex relative to free FVIIa is a critical regulatory point for the coagulation cascade.

FVII is a ~50 kDa, single-chain polypeptide consisting of 406 amino acid residues, with an N-terminal g-carboxyglutamate-rich (GLA) domain, two epidermal growth factor-like domains (EGF1 and EFG2), and a C-terminal serine protease domain. FVII is activated to FVIIa by a specific proteolytic cleavage of the Ile- ¹⁵⁴ -Arg ¹⁵² bond in the short linker region between the EGF2 and the protease domain. This cleavage results in the light and heavy chains being held together by a single disulfide bond of Cys ¹³⁵ and Cys ²⁶² . FVIIa binds phospholipid membrane in a Ca ²⁺ -dependent manner through its N- terminal GLA-domain. Immediately C-terminal to the GLA domain is an aromatic stack and two EGF domains. The aromatic stack connects the GLA to EGF1 domain which binds a single Ca ²⁺ ion. Occupancy of this Ca ²⁺ -binding site increases FVIIa amidolytic activity and tissue factor association. The catalytic triad consist of His ¹⁹³ , Asp ²⁴² , and Ser ³⁴⁴ , and binding of a single Ca ²⁺ ion within the FVIIa protease domain is critical for its catalytic activity. Proteolytic activation of F VII to FVIIa frees the newly formed amino terminus at Ile ¹⁵³ to fold back and be inserted into the activation pocket forming a salt bridge with the carboxylate of Asp ³⁴³ to generate the oxyanion hole. Formation of this salt bridge is critical for FVIIa activity. However, oxyanion hole formation does not occur in free FVIIa upon proteolytic activation. As a result, FVIIa circulates in a zymogen-like state that is poorly recognized by plasma protease inhibitors, allowing it to circulate with a half-life of approximately 90 minutes.

Tissue factor-mediated positioning of the FVIIa active site above the membrane surface is important for FVIIa towards cognate substrates. Free FVIIa adopts a stable, extended structure when bound to the membrane with its active site positioned ~80A above the membrane surface. Upon FVIIa binding to tissue factor, the FVa active site is repositioned ~6A closer to the membrane. This modulation may aid in a proper alignment of the FVIIa catalytic triad with the target substrate cleavage site. Using GLA- domainless FVIIa, it has been shown that the active site was still positioned a similar distance above the membrane, demonstrating that tissue factor is able to fully support FVIIa active site positioning even in the absence of FVIIa-membrane interaction.

Additional data showed that tissue factor supported full FVIIa proteolytic activity as long as the tissue factor extracellular domain was tethered in some way to the membrane surface. However, raising the active site of FVIIa greater than 8qA above the membrane surface greatly reduced the ability of the tissue factor-FVIIa complex to activate FX but did not diminish tissue factor-FVIIa amidolytic activity.

Alanine scanning mutagenesis has been used to assess the role of specific amino acid side chains in the tissue factor extracellular domain for interaction with FVIIa (Gibbs et ak, Biochemistry 33(47): 14003-14010, 1994; Schullek et ah, J Biol Chem 269(30): 19399-19403, 1994). Alanine substitution identified a limited number of residue positions at which alanine replacements cause 5- to lO-fold lower affinity for FVIIa binding. Most of these residue side chains were found to be well-exposed to solvent in the crystal structure, concordant with macromolecular ligand interaction. The FVIIa ligand-binding site is located over an extensive region at the boundary between the two modules. In the C-module, residues Arg ¹³⁵ and Phe ¹⁴⁰ located on the protruding B-C loop provide an independent contact with FVIIa. Leu ¹³³ is located at the base of the fingerlike structure and packed into the cleft between the two modules. This provides continuity to a major cluster of important binding residues consisting of Lys ²⁰ , Thr ⁶⁰ , Asp ⁵⁸ , and Ile ²² . Thr ⁶⁰ is only partially solvent-exposed and may play a local structural role rather than making a significant contact with ligand. The binding site extends onto the concave side of the intermodule angle involving Glu ²⁴ and Gln ¹¹⁰ , and potentially the more distant residue Val ²⁰⁷ . The binding region extends from Asp58 onto a convex surface area formed by Lys ⁴⁸ , Lys ⁴⁶ , Gln ³⁷ , Asp ⁴⁴ , and Trp ⁴⁵ . Trp ⁴⁵ and Asp ⁴⁴ do not interact independently with FVIIa, indicating that the mutational effect at the Trp ⁴⁵ position may reflect a structural importance of this side chain for the local packing of the adjacent Asp ⁴⁴ and Gln ³⁷ side chain. The interactive area further includes two surface- exposed aromatic residues, Phe ⁷⁶ and Tyr ⁷⁸ , which form part of the hydrophobic cluster in the N-module.

The known physiologic substrates of tissue factor-FVIIa are FVII, FIX, and FX and certain proteinase-activated receptors. Mutational analysis has identified a number of residues that, when mutated, support full FVIIa amidolytic activity towards small peptidyl substrates but are deficient in their ability to support macromolecular substrate (i.e., FVII, FIX, and FX) activation (Ruf et ak, J Biol Chem 267(31): 22206-22210, 1992; Ruf et ak, J Biol Chem 267(9): 6375-6381, 1992; Huang et ak, J Biol Chem 271(36): 21752-21757, 1996; Kirchhofer et ak, Biochemistry 39(25): 7380-7387, 2000). The tissue factor loop region at residues 159-165, and residues in or adjacent to this flexible loop have been shown to be critical for the proteolytic activity of the tissue factor-FVIIa complex. This defines the proposed substrate-binding exosite region of tissue factor that is quite distant from the FVIIa active site. A substitution of the glycine residue by a marginally bulkier residue alanine, significantly impairs tissue factor-FVIIa proteolytic activity. This suggests that the flexibility afforded by glycine is critical for the loop of residues 159-165 for tissue factor macromolecular substrate recognition.

The residues Lys ¹⁶⁵ and Lys ¹⁶⁶ have also been demonstrated to be important for substrate recognition and binding. Mutation of either of these residues to alanine results in a significant decrease in the tissue factor co-factor function. Lys ¹⁶⁵ and Lys ¹⁶⁶ face away from each other, with Lys ¹⁶⁵ pointing towards FVIIa in most tissue factor-FVIIa structures, and Lys ¹⁶⁶ pointing into the substrate binding exosite region in the crystal structure. Putative salt bridge formation between Lys ¹⁶⁵ of and Gla ³⁵ of F Vila would support the notion that tissue factor interaction with the GLA domain of F Vila modulates substrate recognition. These results suggest that the C-terminal portion of the tissue factor ectodomain directly interacts with the GLA-domain, the possible adjacent EGF1 domains, of FIX and FX, and that the presence of the FVIIa GLA-domain may modulate these interactions either directly or indirectly.

Exemplary Linker Domains and IgGl Antibody Constructs

In some examples of any of the methods, compositions, or kits described herein, the linker domain can be or include any antigen that is recognized by a cognate IgGl antibody (e.g., a monoclonal antibody). IgGl antibodies are distinguished from other IgG classes of antibodies by their constant region, particularly in hinge regions and upper CH2 domains (see, e.g., Vadarsson et ak, IgG Subclasses and Allotypes: From Structure to Effector Functions, Front Immunol., 5, Article 520, 2014). Human IgGl is the only known IgG subclass which binds to human CDl6a and activates the signaling of human CDl6a.

Any of a variety of linker domains can be used in the single-chain chimeric polypeptides and multi-chain chimeric polypeptides provided herein. In certain embodiments, a linker domain is recognized by an antibody (e.g., an IgGl antibody) or antibody fragment. In some embodiments, the linker domain is a kappa light chain of an antibody that is recognized by a cognate anti-kappa light chain IgGl antibody. In some embodiments, the linker domain is a lambda light chain of an antibody that is recognized by a cognate anti -lambda light chain human IgGl antibody. A variety of kappa light chains and lambda light chains are known in the art (see, e.g., Smith et ak, Antigen Nature and Complexity Influence Human Antibody Light Chain Usage and Specificity, Vaccine, 34(25): 2813-2820, 2016). In some embodiments, the linker domain is or comprises a human polypeptide (e.g., a human kappa light chain of an antibody or a human lambda light chain of an antibody) that is recognized by a cognate human IgGl antibody (e.g., a monoclonal antibody). In some embodiments, the cognate human IgGl antibody includes at least two antigen-binding domains, and each antigen-binding domain binds specifically to the linker domain. In some embodiments, the cognate human IgGl antibody includes at least two antigen-binding domains, and only a subset (e.g., one) of the antigen-binding domains bind specifically to the linker domain. In some

embodiments, a linker domain can be any of the soluble tissue factor domains described herein.

In some embodiments of any of the methods, compositions, and kits described herein, an IgGl antibody construct can be an IgGl antibody (e.g., a monoclonal or a polyclonal IgGl antibody that binds specifically to the linker domain). In some embodiments of any of the methods, compositions, and kits described herein, an IgGl antibody construct can be an antibody or an antibody fragment that includes an IgGl Fc region (e.g., a human IgGl Fc region) and that binds specifically to the linker domain.

As is known in the art, the IgGl Fc region binds to CDl6a (FcRgammalll) (e.g., human CDl6a) and induces its intracellular signaling. In some embodiments, an IgGl antibody construct can be a single chain or a multi-chain polypeptide that includes an Fc region that is capable of binding specifically to CDl6a (FcRgammalll) (e.g., human CDl6a) and is capable of inducing its intracellular signaling in a natural killer cell (e.g., a human natural killer cell), and specifically binds to the linker domain. In some embodiments, an IgGl antibody construct can be a single chain or a multi-chain polypeptide that includes an Fc region that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, or at least 99% identical) to a wildtype IgGl Fc domain (e.g., a wildtype human IgGl Fc domain, e.g., SEQ ID NO: 96) and is capable of binding specifically to CDl6a (FcRgammalll) (e.g., human CDl6a), and is capable of inducing its intracellular signaling in a natural killer cell (e.g., a human natural killer cell), and specifically binds to the linker domain. In some embodiments of any of the methods, compositions, or kits described herein, the IgGl antibody construct can be an antibody or antibody fragment that specifically binds to the linker domain and includes a non-IgGl Fc region (e.g., an IgG2, IgG3, or IgG4 Fc region) that has been altered (e.g., by substituting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in the wildtype non-IgGl Fc region) such that the non-IgGl Fc region is capable of binding to CDl6a (FcRgammalll) (e.g., human CDl6a) and inducing its intracellular signaling in a natural killer cell (e.g., a human natural killer cell). In some embodiments of any of the methods, compositions, or kits described herein, the IgGl antibody construct binds specifically to the linker domain and includes a non-human Fc region (e.g., a Fc region from a non-human antibody) that has been altered (e.g., by substituting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in the wildtype non-human Fc region) such that the non-human Fc region is capable of binding to human CDl6a (human FcRgammalll) and inducing its intracellular signaling in a natural killer cell (e.g., a human natural killer cell).

Wildtype Human IgGl Fc Region (SEQ ID NO: 96)

PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQ

YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQV

SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEA

LHNHYTQKSLSLSPGK

Wildtype Human IgG2 Fc Region (SEQ ID NO: 97)

APPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPR EEQFNST

FRVVSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREE MTKNQVSLTC

LVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNH

YTQKSLSLSPGK

Wildtype Human IgG3 Fc Region (SEQ ID NO: 98)

APELLGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNS TFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMT KNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCS VMHEALHN RFTQKSLSLSPGK

Wildtype Human IgG4 Fc Region (SEQ ID NO: 99)

APEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT KNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHN HYTQKSLSLSLGK In some embodiments, the linker domain can have a total length of about 20 amino acids to about 220 amino acids, about 20 amino acids to about 215 amino acids, about 20 amino acids to about 210 amino acids, about 20 amino acids to about 205 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 195 amino acids, about 20 amino acids to about 190 amino acids, about 20 amino acids to about 185 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 175 amino acids, about 20 amino acids to about 170 amino acids, about 20 amino acids to about 165 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 30 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 215 amino acids, about 30 amino acids to about 210 amino acids, about 30 amino acids to about 205 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, about 30 amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids, about 30 amino acids to about 110 amino acids, about 30 amino acids to about 105 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 95 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 75 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 40 amino acids, about 40 amino acids to about 220 amino acids, about 40 amino acids to about 215 amino acids, about 40 amino acids to about 210 amino acids, about 40 amino acids to about 205 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 215 amino acids, about 50 amino acids to about 210 amino acids, about 50 amino acids to about 205 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 145 amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 75 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 220 amino acids, about 60 amino acids to about 215 amino acids, about 60 amino acids to about 210 amino acids, about 60 amino acids to about 205 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 195 amino acids, about 60 amino acids to about 190 amino acids, about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 220 amino acids, about 70 amino acids to about 215 amino acids, about 70 amino acids to about 210 amino acids, about 70 amino acids to about 205 amino acids, about 70 amino acids to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 220 amino acids, about 80 amino acids to about 215 amino acids, about 80 amino acids to about 210 amino acids, about 80 amino acids to about 205 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 175 amino acids, about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 215 amino acids, about 90 amino acids to about 210 amino acids, about 90 amino acids to about 205 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 215 amino acids, about 100 amino acids to about 210 amino acids, about 100 amino acids to about 205 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 215 amino acids, about 110 amino acids to about 210 amino acids, about 110 amino acids to about 205 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 220 amino acids, about 115 amino acids to about 215 amino acids, about 115 amino acids to about 210 amino acids, about 115 amino acids to about 205 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 215 amino acids, about 120 amino acids to about 210 amino acids, about 120 amino acids to about 205 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to about 185 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 175 amino acids, about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to about 150 amino acids, about 120 amino acids to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 215 amino acids, about 125 amino acids to about 210 amino acids, about 125 amino acids to about 205 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids, about 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 220 amino acids, about 130 amino acids to about 215 amino acids, about 130 amino acids to about 210 amino acids, about 130 amino acids to about 205 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 220 amino acids, about 135 amino acids to about 215 amino acids, about 135 amino acids to about 210 amino acids, about 135 amino acids to about 205 amino acids, about 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids, about 135 amino acids to about 140 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 215 amino acids, about 140 amino acids to about 210 amino acids, about 140 amino acids to about 205 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140 amino acids to about 145 amino acids, about 145 amino acids to about 220 amino acids, about 145 amino acids to about 215 amino acids, about 145 amino acids to about 210 amino acids, about 145 amino acids to about 205 amino acids, about 145 amino acids to about 200 amino acids, about 145 amino acids to about 195 amino acids, about 145 amino acids to about 190 amino acids, about 145 amino acids to about 185 amino acids, about 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 215 amino acids, about 150 amino acids to about 210 amino acids, about 150 amino acids to about 205 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 215 amino acids, about 155 amino acids to about 210 amino acids, about 155 amino acids to about 205 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 215 amino acids, about 160 amino acids to about 210 amino acids, about 160 amino acids to about 205 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 amino acids to about 190 amino acids, about 160 amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids, about 165 amino acids to about 220 amino acids, about 165 amino acids to about 215 amino acids, about 165 amino acids to about 210 amino acids, about 165 amino acids to about 205 amino acids, about 165 amino acids to about 200 amino acids, about 165 amino acids to about 195 amino acids, about 165 amino acids to about 190 amino acids, about 165 amino acids to about 185 amino acids, about 165 amino acids to about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 215 amino acids, about 170 amino acids to about 210 amino acids, about 170 amino acids to about 205 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 220 amino acids, about 175 amino acids to about 215 amino acids, about 175 amino acids to about 210 amino acids, about 175 amino acids to about 205 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to about 190 amino acids, about 175 amino acids to about 185 amino acids, about 175 amino acids to about 180 amino acids, about 180 amino acids to about 220 amino acids, about 180 amino acids to about 215 amino acids, about 180 amino acids to about 210 amino acids, about 180 amino acids to about 205 amino acids, about 180 amino acids to about 200 amino acids, about 180 amino acids to about 195 amino acids, about 180 amino acids to about 190 amino acids, about 180 amino acids to about 185 amino acids, about 185 amino acids to about 220 amino acids, about 185 amino acids to about 215 amino acids, about 185 amino acids to about 210 amino acids, about 185 amino acids to about 205 amino acids, about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195 amino acids, about 185 amino acids to about 190 amino acids, about 190 amino acids to about 220 amino acids, about 190 amino acids to about 215 amino acids, about 190 amino acids to about 210 amino acids, about 190 amino acids to about 205 amino acids, about 190 amino acids to about 200 amino acids, about 190 amino acids to about 195 amino acids, about 195 amino acids to about 220 amino acids, about 195 amino acids to about 215 amino acids, about 195 amino acids to about 210 amino acids, about 195 amino acids to about 205 amino acids, about 195 amino acids to about 200 amino acids, about 200 amino acids to about 220 amino acids, about 200 amino acids to about 215 amino acids, about 200 amino acids to about 210 amino acids, about 200 amino acids to about 205 amino acids, about 205 amino acids to about 220 amino acids, about 205 amino acids to about 215 amino acids, about 205 amino acids to about 210 amino acids, about 210 amino acids to about 220 amino acids, about 210 amino acids to about 215 amino acids, or about 215 amino acids to about 220 amino acids. Soluble Tissue Factor Domain

In some examples of any of the methods, compositions, or kits described herein, the linker domain can be a soluble tissue factor domain. In some embodiments, the soluble tissue factor domain can be a wildtype tissue factor polypeptide lacking the signal sequence, the transmembrane domain, and the intracellular domain. In some examples, the soluble tissue factor domain can be a tissue factor mutant, wherein a wildtype tissue factor polypeptide lacking the signal sequence, the transmembrane domain, and the intracellular domain, and has been further modified at selected amino acids. In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble rat tissue factor domain. Non-limiting examples of soluble human tissue factor domains, a mouse soluble tissue factor domain, a rat soluble tissue factor domain, and mutant soluble tissue factor domains are shown below. Exemplary Soluble Human Tissue Factor Domain (SEQ ID NO: 1)

S GTTNT VA AYNLT WK S TNFKTILEWEPKP VN Q V YT V QI S TK S GD WK SKCF YTTD TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQ PTIQ SFEQ V GTK VN VT VEDERTLVRRNNTFL SLRD VF GKDLI YTLYYWK S S S S GK KTAKTNTNEFLID VDKGEN Y CF S VQ AVIP SRT VNRK S TD SP VECMGQEKGEFRE

Exemplary Nucleic Acid Encoding Soluble Human Tissue Factor Domain (SEQ ID NO: 2)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA

ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACC

GTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCAC

CGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACC

TACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTC

CGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGA

CCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGT

GAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTT

CTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGG

AAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTT

TAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATC

CCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGG

GCCAAGAAAAGGGCGAGTTCCGGGAG

Exemplary Soluble Mouse Tissue Factor Domain (SEQ ID NO: 3)

AGI PEKAFNLTWISTDFKTILEWQPKPTNYTYTVQISDRSRNWKNKCFSTT DTECDLTDEIVKDVTWAYEAKVLSVPRRNSVHGDGDQLVIHGEEPPFTNAP KFLPYRDTNLGQPVIQQFEQDGRKLNVWKDSLTLVRKNGTFLTLRQVFGK DLGYI ITYRKGSSTGKKTNITNTNEFSIDVEEGVSYCFFVQAMI FSRKTNQ NSPGSSTVCTEQWKSFLGE Exemplary Soluble Rat Tissue Factor Domain (SEQ ID NO: 4)

AGTPPGKAFNLTWISTDFKTILEWQPKPTNYTYTVQISDRSRNWKYKCTGT TDTECDLTDEIVKDVNWTYEARVLSVPWRNSTHGKETLFGTHGEEPPFTNA RKFLPYRDTKIGQPVIQKYEQGGTKLKVTVKDSFTLVRKNGTFLTLRQVFG NDLGYILTYRKDSSTGRKTNTTHTNEFLIDVEKGVSYCFFAQAVI FSRKTN HKSPESITKCTEQWKSVLGE

Exemplary Mutant Soluble Human Tissue Factor Domain (SEQ ID NO: 5)

S GTTNT V A A YNLTWK S TNF AT ALEWEPKP VN Q V YT V QI S TK S GD WK SKCF YTT DTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNL GQPTIQ SFEQ VGTKVN VTVEDERTL VARNNT AL SLRD VFGKDLI YTL YYWKS S S S GKKT AKTNTNEFLID VDKGEN Y CF S VQ A VIP SRT VNRK S TD SP VECMGQEKGEF RE

Exemplary Mutant Soluble Human Tissue Factor Domain (SEQ ID NO: 6)

S GTTNT V A A YNLTWK S TNF AT ALEWEPKP VN Q V YT V QI S TK S GD AK SKCF YTTD

TECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLG QPTIQ SFEQ V GTKVN VTVEDERTL VARNNT AL SLRD VF GKDLI YTL YYWK S S S S G KKT AKTNTNEFLID VDKGEN Y CF S VQ A VIP SRT VNRK S TD SP VECMGQEKGEFR E

In some embodiments, a soluble tissue factor domain can include a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 1, 3, 4, 5, or 6. In some embodiments, a soluble tissue factor domain can include a sequence of SEQ ID NO: 1, 3, 4, 5, or 6 with one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids removed from its N-terminus and/or one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids removed from its C-terminus.

As will be appreciated by those of skill in the art, mutation of amino acids that are conserved between different mammalian species is more likely to decrease the activity and/or structural stability of the protein, while mutation of amino acids that are not conserved between different mammalian species is less likely to decrease the activity and/or structural stability of the protein.

In some examples, the soluble tissue factor domain is not capable of binding to Factor Vila. In some examples, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments, the single-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble rat tissue factor domain.

In some examples, the soluble tissue factor domain does not include one or more (e.g., two, three, four, five, six or seven) of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

In some embodiments, the mutant soluble tissue factor possesses the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

In some examples, the soluble tissue factor domain can be encoded by a nucleic acid including a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 2.

In some embodiments, the soluble tissue factor domain can have a total length of about 20 amino acids to about 220 amino acids, about 20 amino acids to about 215 amino acids, about 20 amino acids to about 210 amino acids, about 20 amino acids to about 205 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 195 amino acids, about 20 amino acids to about 190 amino acids, about 20 amino acids to about 185 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 175 amino acids, about 20 amino acids to about 170 amino acids, about 20 amino acids to about 165 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 30 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 215 amino acids, about 30 amino acids to about 210 amino acids, about 30 amino acids to about 205 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, about 30 amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids, about 30 amino acids to about 110 amino acids, about 30 amino acids to about 105 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 95 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 75 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 40 amino acids, about 40 amino acids to about 220 amino acids, about 40 amino acids to about 215 amino acids, about 40 amino acids to about 210 amino acids, about 40 amino acids to about 205 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 215 amino acids, about 50 amino acids to about 210 amino acids, about 50 amino acids to about 205 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 145 amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 75 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 220 amino acids, about 60 amino acids to about 215 amino acids, about 60 amino acids to about 210 amino acids, about 60 amino acids to about 205 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 195 amino acids, about 60 amino acids to about 190 amino acids, about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 220 amino acids, about 70 amino acids to about 215 amino acids, about 70 amino acids to about 210 amino acids, about 70 amino acids to about 205 amino acids, about 70 amino acids to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 220 amino acids, about 80 amino acids to about 215 amino acids, about 80 amino acids to about 210 amino acids, about 80 amino acids to about 205 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 175 amino acids, about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 215 amino acids, about 90 amino acids to about 210 amino acids, about 90 amino acids to about 205 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 215 amino acids, about 100 amino acids to about 210 amino acids, about 100 amino acids to about 205 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 215 amino acids, about 110 amino acids to about 210 amino acids, about 110 amino acids to about 205 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 220 amino acids, about 115 amino acids to about 215 amino acids, about 115 amino acids to about 210 amino acids, about 115 amino acids to about 205 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 215 amino acids, about 120 amino acids to about 210 amino acids, about 120 amino acids to about 205 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to about 185 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 175 amino acids, about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to about 150 amino acids, about 120 amino acids to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 215 amino acids, about 125 amino acids to about 210 amino acids, about 125 amino acids to about 205 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids, about 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 220 amino acids, about 130 amino acids to about 215 amino acids, about 130 amino acids to about 210 amino acids, about 130 amino acids to about 205 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 220 amino acids, about 135 amino acids to about 215 amino acids, about 135 amino acids to about 210 amino acids, about 135 amino acids to about 205 amino acids, about 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids, about 135 amino acids to about 140 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 215 amino acids, about 140 amino acids to about 210 amino acids, about 140 amino acids to about 205 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140 amino acids to about 145 amino acids, about 145 amino acids to about 220 amino acids, about 145 amino acids to about 215 amino acids, about 145 amino acids to about 210 amino acids, about 145 amino acids to about 205 amino acids, about 145 amino acids to about 200 amino acids, about 145 amino acids to about 195 amino acids, about 145 amino acids to about 190 amino acids, about 145 amino acids to about 185 amino acids, about 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 215 amino acids, about 150 amino acids to about 210 amino acids, about 150 amino acids to about 205 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 215 amino acids, about 155 amino acids to about 210 amino acids, about 155 amino acids to about 205 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 215 amino acids, about 160 amino acids to about 210 amino acids, about 160 amino acids to about 205 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 amino acids to about 190 amino acids, about 160 amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids, about 165 amino acids to about 220 amino acids, about 165 amino acids to about 215 amino acids, about 165 amino acids to about 210 amino acids, about 165 amino acids to about 205 amino acids, about 165 amino acids to about 200 amino acids, about 165 amino acids to about 195 amino acids, about 165 amino acids to about 190 amino acids, about 165 amino acids to about 185 amino acids, about 165 amino acids to about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 215 amino acids, about 170 amino acids to about 210 amino acids, about 170 amino acids to about 205 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 220 amino acids, about 175 amino acids to about 215 amino acids, about 175 amino acids to about 210 amino acids, about 175 amino acids to about 205 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to about 190 amino acids, about 175 amino acids to about 185 amino acids, about 175 amino acids to about 180 amino acids, about 180 amino acids to about 220 amino acids, about 180 amino acids to about 215 amino acids, about 180 amino acids to about 210 amino acids, about 180 amino acids to about 205 amino acids, about 180 amino acids to about 200 amino acids, about 180 amino acids to about 195 amino acids, about 180 amino acids to about 190 amino acids, about 180 amino acids to about 185 amino acids, about 185 amino acids to about 220 amino acids, about 185 amino acids to about 215 amino acids, about 185 amino acids to about 210 amino acids, about 185 amino acids to about 205 amino acids, about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195 amino acids, about 185 amino acids to about 190 amino acids, about 190 amino acids to about 220 amino acids, about 190 amino acids to about 215 amino acids, about 190 amino acids to about 210 amino acids, about 190 amino acids to about 205 amino acids, about 190 amino acids to about 200 amino acids, about 190 amino acids to about 195 amino acids, about 195 amino acids to about 220 amino acids, about 195 amino acids to about 215 amino acids, about 195 amino acids to about 210 amino acids, about 195 amino acids to about 205 amino acids, about 195 amino acids to about 200 amino acids, about 200 amino acids to about 220 amino acids, about 200 amino acids to about 215 amino acids, about 200 amino acids to about 210 amino acids, about 200 amino acids to about 205 amino acids, about 205 amino acids to about 220 amino acids, about 205 amino acids to about 215 amino acids, about 205 amino acids to about 210 amino acids, about 210 amino acids to about 220 amino acids, about 210 amino acids to about 215 amino acids, or about 215 amino acids to about 220 amino acids.

Linker Sequences

In some embodiments, the linker sequence can be a flexible linker sequence. Non-limiting examples of linker sequences that can be used are described in Klein et ah, Protein Engineering, Design & Selection Vol. 27, No. 10, pp. 325-330, 2014; Priyanka et ah, Protein Sci., 2013 Feb; 22(2): 153-167. In some examples, the linker sequence is a synthetic linker sequence.

In some embodiments of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein, the polypeptide can include one, two, three, four, five, six, seven, eight, nine, or ten linker sequence(s) (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art). In some embodiments of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein, the polypeptide can include one, two, three, four, five, six, seven, eight, nine, or ten linker sequence(s) (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art).

In some embodiments, a linker sequence can have a total length of 1 amino acid to about 100 amino acids, 1 amino acid to about 90 amino acids, 1 amino acid to about 80 amino acids, 1 amino acid to about 70 amino acids, 1 amino acid to about 60 amino acids, 1 amino acid to about 50 amino acids, 1 amino acid to about 45 amino acids, 1 amino acid to about 40 amino acids, 1 amino acid to about 35 amino acids, 1 amino acid to about 30 amino acids, 1 amino acid to about 25 amino acids, 1 amino acid to about 24 amino acids, 1 amino acid to about 22 amino acids, 1 amino acid to about 20 amino acids,

1 amino acid to about 18 amino acids, 1 amino acid to about 16 amino acids, 1 amino acid to about 14 amino acids, 1 amino acid to about 12 amino acids, 1 amino acid to about 10 amino acids, 1 amino acid to about 8 amino acids, 1 amino acid to about 6 amino acids, 1 amino acid to about 4 amino acids, about 2 amino acids to about 100 amino acids, about 2 amino acids to about 90 amino acids, about 2 amino acids to about 80 amino acids, about 2 amino acids to about 70 amino acids, about 2 amino acids to about 60 amino acids, about 2 amino acids to about 50 amino acids, about 2 amino acids to about 45 amino acids, about 2 amino acids to about 40 amino acids, about 2 amino acids to about 35 amino acids, about 2 amino acids to about 30 amino acids, about 2 amino acids to about 25 amino acids, about 2 amino acids to about 24 amino acids, about

2 amino acids to about 22 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 18 amino acids, about 2 amino acids to about 16 amino acids, about 2 amino acids to about 14 amino acids, about 2 amino acids to about 12 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 4 amino acids, about 4 amino acids to about 100 amino acids, about 4 amino acids to about

90 amino acids, about 4 amino acids to about 80 amino acids, about 4 amino acids to about 70 amino acids, about 4 amino acids to about 60 amino acids, about 4 amino acids to about 50 amino acids, about 4 amino acids to about 45 amino acids, about 4 amino acids to about 40 amino acids, about 4 amino acids to about 35 amino acids, about 4 amino acids to about 30 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 24 amino acids, about 4 amino acids to about 22 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 18 amino acids, about 4 amino acids to about 16 amino acids, about 4 amino acids to about 14 amino acids, about 4 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 6 amino acids, about 6 amino acids to about 100 amino acids, about 6 amino acids to about 90 amino acids, about 6 amino acids to about 80 amino acids, about 6 amino acids to about 70 amino acids, about 6 amino acids to about 60 amino acids, about 6 amino acids to about 50 amino acids, about 6 amino acids to about 45 amino acids, about 6 amino acids to about 40 amino acids, about 6 amino acids to about 35 amino acids, about 6 amino acids to about 30 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 24 amino acids, about 6 amino acids to about 22 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 18 amino acids, about 6 amino acids to about 16 amino acids, about 6 amino acids to about 14 amino acids, about 6 amino acids to about 12 amino acids, about 6 amino acids to about 10 amino acids, about 6 amino acids to about 8 amino acids, about 8 amino acids to about 100 amino acids, about 8 amino acids to about 90 amino acids, about 8 amino acids to about 80 amino acids, about 8 amino acids to about 70 amino acids, about 8 amino acids to about 60 amino acids, about 8 amino acids to about 50 amino acids, about 8 amino acids to about 45 amino acids, about 8 amino acids to about 40 amino acids, about 8 amino acids to about 35 amino acids, about 8 amino acids to about 30 amino acids, about 8 amino acids to about 25 amino acids, about 8 amino acids to about 24 amino acids, about 8 amino acids to about 22 amino acids, about 8 amino acids to about 20 amino acids, about 8 amino acids to about 18 amino acids, about 8 amino acids to about 16 amino acids, about 8 amino acids to about 14 amino acids, about 8 amino acids to about 12 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 24 amino acids, about 10 amino acids to about 22 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 18 amino acids, about 10 amino acids to about 16 amino acids, about 10 amino acids to about 14 amino acids, about 10 amino acids to about 12 amino acids, about 12 amino acids to about 100 amino acids, about 12 amino acids to about 90 amino acids, about 12 amino acids to about 80 amino acids, about 12 amino acids to about 70 amino acids, about 12 amino acids to about 60 amino acids, about 12 amino acids to about 50 amino acids, about 12 amino acids to about 45 amino acids, about 12 amino acids to about 40 amino acids, about 12 amino acids to about 35 amino acids, about 12 amino acids to about 30 amino acids, about 12 amino acids to about 25 amino acids, about 12 amino acids to about 24 amino acids, about 12 amino acids to about 22 amino acids, about 12 amino acids to about 20 amino acids, about 12 amino acids to about 18 amino acids, about 12 amino acids to about 16 amino acids, about 12 amino acids to about 14 amino acids, about 14 amino acids to about 100 amino acids, about 14 amino acids to about 90 amino acids, about 14 amino acids to about 80 amino acids, about 14 amino acids to about 70 amino acids, about 14 amino acids to about 60 amino acids, about 14 amino acids to about 50 amino acids, about 14 amino acids to about 45 amino acids, about 14 amino acids to about 40 amino acids, about 14 amino acids to about 35 amino acids, about 14 amino acids to about 30 amino acids, about 14 amino acids to about 25 amino acids, about 14 amino acids to about 24 amino acids, about 14 amino acids to about 22 amino acids, about 14 amino acids to about 20 amino acids, about 14 amino acids to about 18 amino acids, about 14 amino acids to about 16 amino acids, about 16 amino acids to about 100 amino acids, about 16 amino acids to about 90 amino acids, about 16 amino acids to about 80 amino acids, about 16 amino acids to about 70 amino acids, about 16 amino acids to about 60 amino acids, about 16 amino acids to about 50 amino acids, about 16 amino acids to about 45 amino acids, about 16 amino acids to about 40 amino acids, about 16 amino acids to about 35 amino acids, about 16 amino acids to about 30 amino acids, about 16 amino acids to about 25 amino acids, about 16 amino acids to about 24 amino acids, about 16 amino acids to about 22 amino acids, about 16 amino acids to about 20 amino acids, about 16 amino acids to about 18 amino acids, about 18 amino acids to about 100 amino acids, about 18 amino acids to about 90 amino acids, about 18 amino acids to about 80 amino acids, about 18 amino acids to about 70 amino acids, about 18 amino acids to about 60 amino acids, about 18 amino acids to about 50 amino acids, about 18 amino acids to about 45 amino acids, about 18 amino acids to about 40 amino acids, about 18 amino acids to about 35 amino acids, about 18 amino acids to about 30 amino acids, about 18 amino acids to about 25 amino acids, about 18 amino acids to about 24 amino acids, about 18 amino acids to about 22 amino acids, about 18 amino acids to about 20 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 20 amino acids to about 24 amino acids, about 20 amino acids to about 22 amino acids, about 22 amino acids to about 100 amino acids, about 22 amino acids to about 90 amino acids, about 22 amino acids to about 80 amino acids, about 22 amino acids to about 70 amino acids, about 22 amino acids to about 60 amino acids, about 22 amino acids to about 50 amino acids, about 22 amino acids to about 45 amino acids, about 22 amino acids to about 40 amino acids, about 22 amino acids to about 35 amino acids, about 22 amino acids to about 30 amino acids, about 22 amino acids to about 25 amino acids, about 22 amino acids to about 24 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 80 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 80 amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to about 60 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 90 amino acids, or about 90 amino acids to about 100 amino acids.

In some embodiments, the linker sequence is rich in glycine (Gly or G) residues.

In some embodiments, the linker sequence is rich in serine (Ser or S) residues. In some embodiments, the linker sequence is rich in glycine and serine residues. In some embodiments, the linker sequence has one or more glycine-serine residue pairs (GS), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. In some embodiments, the linker sequence has one or more Gly-Gly-Gly-Ser (GGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences. In some embodiments, the linker sequence has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences. In some embodiments, the linker sequence has one or more Gly- Gly-Ser-Gly (GGSG) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences.

In some embodiments, the linker sequence can comprise or consist of

GGGGSGGGGSGGGGS (SEQ ID NO: 7). In some embodiments, the linker sequence can be encoded by a nucleic acid comprising or consisting of:

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO: 8). In some embodiments, the linker sequence can comprise or consist of:

GGGSGGGS (SEQ ID NO: 9). Target-Binding Domains

In some embodiments of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein, the first target-binding domain, the second target-binding domain, and/or the additional one or more target-binding domains can be an antigen-binding domain (e.g., any of the exemplary antigen-binding domains described herein or known in the art), a soluble interleukin or cytokine protein (e.g., any of the exemplary soluble interleukin proteins or soluble cytokine proteins described herein or known in the art), a soluble interleukin or cytokine receptor (e.g., any of the exemplary soluble interleukin receptors or soluble cytokine receptors described herein or known in the art), and ligands of co-stimulatory molecules (e.g., any of the exemplary ligands of co-stimulatory molecules described herein or known in the art).

In some embodiments of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary first target binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary second target binding domains described herein or known in the art), and the one or more additional target binding domains can each, independently, bind specifically to a target selected from the group of: CDl6a, CD28, CD3 (e.g., one or more of CD3a, CD3P, CD36, CD3e, and CD3y), CD33, CD20, CD 19, CD22, CD40, CD47, CD52, CD70, CD80, CD86, CD123, CD137, CD272, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, PDL-2, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, B7-H4, HVEM, EPCAM, BCMA, P-cadherin, CEACAM5, VISTA, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER,

CD 122, CD155, PDGF-DD, ILT3, ILT4, TIGIT, MHCII, LAG3, 0X40, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF- b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a EILP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD28.

In some embodiments of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein, the first target-binding domain, the second target-binding domain, and/or the one or more additional target-binding domains, can be a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. In some examples, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL- 12, IL-l 5, IL-l 7, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16- binding protein.

In some embodiments of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein, the first target-binding domain, the second target-binding domain, and/or the one or more additional target-binding domains, can be a soluble interleukin receptor, a soluble cytokine receptor, or a soluble cell surface receptor. In some examples, the soluble interleukin receptor, the soluble cytokine receptor, or the soluble cell surface receptor is soluble TGF-b receptor II (TGF- b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28.

In some embodiments of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein, the first target-binding domain, the second target-binding domain, and/or the one or more additional target-binding domains, can be a ligand of a co-stimulatory molecule. Non-limiting examples of ligands of a co-stimulatory molecule include soluble CD80, soluble CD86, soluble CD40, soluble ICOSL, soluble CD70, soluble OX40L, soluble 4-1BBL, soluble GITRL, soluble LIGHT, soluble TIM3, soluble TIM4, soluble ICAM1, soluble LFA3, soluble CDld, or soluble LLT-l.

In some embodiments of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein, the first target-binding domain, the second target-binding domain, and/or the one or more additional target-binding domains can each independent have a total number of amino acids of about 5 amino acids to about 1000 amino acids, about 5 amino acids to about 950 amino acids, about 5 amino acids to about 900 amino acids, about 5 amino acids to about 850 amino acids, about 5 amino acids to about 800 amino acids, about 5 amino acids to about 750 amino acids, about 5 amino acids to about 700 amino acids, about 5 amino acids to about 650 amino acids, about 5 amino acids to about 600 amino acids, about 5 amino acids to about 550 amino acids, about 5 amino acids to about 500 amino acids, about 5 amino acids to about 450 amino acids, about 5 amino acids to about 400 amino acids, about 5 amino acids to about 350 amino acids, about 5 amino acids to about 300 amino acids, about 5 amino acids to about 280 amino acids, about 5 amino acids to about 260 amino acids, about 5 amino acids to about 240 amino acids, about 5 amino acids to about 220 amino acids, about 5 amino acids to about 200 amino acids, about 5 amino acids to about 195 amino acids, about 5 amino acids to about 190 amino acids, about 5 amino acids to about 185 amino acids, about 5 amino acids to about 180 amino acids, about 5 amino acids to about 175 amino acids, about 5 amino acids to about 170 amino acids, about 5 amino acids to about 165 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 155 amino acids, about 5 amino acids to about 150 amino acids, about 5 amino acids to about 145 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 135 amino acids, about 5 amino acids to about 130 amino acids, about 5 amino acids to about 125 amino acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 115 amino acids, about 5 amino acids to about 110 amino acids, about 5 amino acids to about 105 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 95 amino acids, about 5 amino acids to about 90 amino acids, about 5 amino acids to about 85 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 75 amino acids, about 5 amino acids to about 70 amino acids, about 5 amino acids to about 65 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 55 amino acids, about 5 amino acids to about 50 amino acids, about 5 amino acids to about 45 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 35 amino acids, about 5 amino acids to about 30 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to about 10 amino acids, about 10 amino acids to about 1000 amino acids, about 10 amino acids to about 950 amino acids, about 10 amino acids to about 900 amino acids, about 10 amino acids to about 850 amino acids, about 10 amino acids to about 800 amino acids, about 10 amino acids to about 750 amino acids, about 10 amino acids to about 700 amino acids, about 10 amino acids to about 650 amino acids, about 10 amino acids to about 600 amino acids, about 10 amino acids to about 550 amino acids, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 450 amino acids, about 10 amino acids to about 400 amino acids, about 10 amino acids to about 350 amino acids, about 10 amino acids to about 300 amino acids, about 10 amino acids to about 280 amino acids, about 10 amino acids to about 260 amino acids, about 10 amino acids to about 240 amino acids, about 10 amino acids to about 220 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 195 amino acids, about 10 amino acids to about 190 amino acids, about 10 amino acids to about 185 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 175 amino acids, about 10 amino acids to about 170 amino acids, about 10 amino acids to about 165 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 155 amino acids, about 10 amino acids to about 150 amino acids, about 10 amino acids to about 145 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 135 amino acids, about 10 amino acids to about 130 amino acids, about 10 amino acids to about 125 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 1 15 amino acids, about 10 amino acids to about 110 amino acids, about 10 amino acids to about 105 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 95 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 85 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 75 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 65 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 55 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 1000 amino acids, about 15 amino acids to about 950 amino acids, about 15 amino acids to about 900 amino acids, about 15 amino acids to about 850 amino acids, about 15 amino acids to about 800 amino acids, about 15 amino acids to about 750 amino acids, about 15 amino acids to about 700 amino acids, about 15 amino acids to about 650 amino acids, about 15 amino acids to about 600 amino acids, about 15 amino acids to about 550 amino acids, about 15 amino acids to about 500 amino acids, about 15 amino acids to about 450 amino acids, about 15 amino acids to about 400 amino acids, about 15 amino acids to about 350 amino acids, about 15 amino acids to about 300 amino acids, about 15 amino acids to about 280 amino acids, about 15 amino acids to about 260 amino acids, about 15 amino acids to about 240 amino acids, about 15 amino acids to about 220 amino acids, about 15 amino acids to about 200 amino acids, about 15 amino acids to about 195 amino acids, about 15 amino acids to about 190 amino acids, about 15 amino acids to about 185 amino acids, about 15 amino acids to about 180 amino acids, about 15 amino acids to about 175 amino acids, about 15 amino acids to about 170 amino acids, about 15 amino acids to about 165 amino acids, about 15 amino acids to about 160 amino acids, about 15 amino acids to about 155 amino acids, about 15 amino acids to about 150 amino acids, about 15 amino acids to about 145 amino acids, about 15 amino acids to about 140 amino acids, about 15 amino acids to about 135 amino acids, about 15 amino acids to about 130 amino acids, about 15 amino acids to about 125 amino acids, about 15 amino acids to about 120 amino acids, about 15 amino acids to about 115 amino acids, about 15 amino acids to about 110 amino acids, about 15 amino acids to about 105 amino acids, about 15 amino acids to about 100 amino acids, about 15 amino acids to about 95 amino acids, about 15 amino acids to about 90 amino acids, about 15 amino acids to about 85 amino acids, about 15 amino acids to about 80 amino acids, about 15 amino acids to about 75 amino acids, about 15 amino acids to about 70 amino acids, about 15 amino acids to about 65 amino acids, about 15 amino acids to about 60 amino acids, about 15 amino acids to about 55 amino acids, about 15 amino acids to about 50 amino acids, about 15 amino acids to about 45 amino acids, about 15 amino acids to about 40 amino acids, about 15 amino acids to about 35 amino acids, about 15 amino acids to about 30 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 20 amino acids, about 20 amino acids to about 1000 amino acids, about 20 amino acids to about 950 amino acids, about 20 amino acids to about 900 amino acids, about 20 amino acids to about 850 amino acids, about 20 amino acids to about 800 amino acids, about 20 amino acids to about 750 amino acids, about 20 amino acids to about 700 amino acids, about 20 amino acids to about 650 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 550 amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 450 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 350 amino acids, about 20 amino acids to about 300 amino acids, about 20 amino acids to about 280 amino acids, about 20 amino acids to about 260 amino acids, about 20 amino acids to about 240 amino acids, about 20 amino acids to about 220 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 195 amino acids, about 20 amino acids to about 190 amino acids, about 20 amino acids to about 185 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 175 amino acids, about 20 amino acids to about 170 amino acids, about 20 amino acids to about 165 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 65 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 55 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 25 amino acids to about 1000 amino acids, about 25 amino acids to about 950 amino acids, about 25 amino acids to about 900 amino acids, about 25 amino acids to about 850 amino acids, about 25 amino acids to about 800 amino acids, about 25 amino acids to about 750 amino acids, about 25 amino acids to about 700 amino acids, about 25 amino acids to about 650 amino acids, about 25 amino acids to about 600 amino acids, about 25 amino acids to about 550 amino acids, about 25 amino acids to about 500 amino acids, about 25 amino acids to about 450 amino acids, about 25 amino acids to about 400 amino acids, about 25 amino acids to about 350 amino acids, about 25 amino acids to about 300 amino acids, about 25 amino acids to about 280 amino acids, about 25 amino acids to about 260 amino acids, about 25 amino acids to about 240 amino acids, about 25 amino acids to about 220 amino acids, about 25 amino acids to about 200 amino acids, about 25 amino acids to about 195 amino acids, about 25 amino acids to about 190 amino acids, about 25 amino acids to about 185 amino acids, about 25 amino acids to about 180 amino acids, about 25 amino acids to about 175 amino acids, about 25 amino acids to about 170 amino acids, about 25 amino acids to about 165 amino acids, about 25 amino acids to about 160 amino acids, about 25 amino acids to about 155 amino acids, about 25 amino acids to about 150 amino acids, about 25 amino acids to about 145 amino acids, about 25 amino acids to about 140 amino acids, about 25 amino acids to about 135 amino acids, about 25 amino acids to about 130 amino acids, about 25 amino acids to about 125 amino acids, about 25 amino acids to about 120 amino acids, about 25 amino acids to about 115 amino acids, about 25 amino acids to about 110 amino acids, about 25 amino acids to about 105 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 95 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 85 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 75 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 65 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 55 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 1000 amino acids, about 30 amino acids to about 950 amino acids, about 30 amino acids to about 900 amino acids, about 30 amino acids to about 850 amino acids, about 30 amino acids to about 800 amino acids, about 30 amino acids to about 750 amino acids, about 30 amino acids to about 700 amino acids, about 30 amino acids to about 650 amino acids, about 30 amino acids to about 600 amino acids, about 30 amino acids to about 550 amino acids, about 30 amino acids to about 500 amino acids, about 30 amino acids to about 450 amino acids, about 30 amino acids to about 400 amino acids, about 30 amino acids to about 350 amino acids, about 30 amino acids to about 300 amino acids, about 30 amino acids to about 280 amino acids, about 30 amino acids to about 260 amino acids, about 30 amino acids to about 240 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, about 30 amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids, about 30 amino acids to about 110 amino acids, about 30 amino acids to about 105 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 95 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 75 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 65 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 55 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 1000 amino acids, about 35 amino acids to about 950 amino acids, about 35 amino acids to about 900 amino acids, about 35 amino acids to about 850 amino acids, about 35 amino acids to about 800 amino acids, about 35 amino acids to about 750 amino acids, about 35 amino acids to about 700 amino acids, about 35 amino acids to about 650 amino acids, about 35 amino acids to about 600 amino acids, about 35 amino acids to about 550 amino acids, about 35 amino acids to about 500 amino acids, about 35 amino acids to about 450 amino acids, about 35 amino acids to about 400 amino acids, about 35 amino acids to about 350 amino acids, about 35 amino acids to about 300 amino acids, about 35 amino acids to about 280 amino acids, about 35 amino acids to about 260 amino acids, about 35 amino acids to about 240 amino acids, about 35 amino acids to about 220 amino acids, about 35 amino acids to about 200 amino acids, about 35 amino acids to about 195 amino acids, about 35 amino acids to about 190 amino acids, about 35 amino acids to about 185 amino acids, about 35 amino acids to about 180 amino acids, about 35 amino acids to about 175 amino acids, about 35 amino acids to about 170 amino acids, about 35 amino acids to about 165 amino acids, about 35 amino acids to about 160 amino acids, about 35 amino acids to about 155 amino acids, about 35 amino acids to about 150 amino acids, about 35 amino acids to about 145 amino acids, about 35 amino acids to about 140 amino acids, about 35 amino acids to about 135 amino acids, about 35 amino acids to about 130 amino acids, about 35 amino acids to about 125 amino acids, about 35 amino acids to about 120 amino acids, about 35 amino acids to about 115 amino acids, about 35 amino acids to about 110 amino acids, about 35 amino acids to about 105 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 95 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 85 amino acids, about 35 amino acids to about 80 amino acids, about 35 amino acids to about 75 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 65 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 55 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 1000 amino acids, about 40 amino acids to about 950 amino acids, about 40 amino acids to about 900 amino acids, about 40 amino acids to about 850 amino acids, about 40 amino acids to about 800 amino acids, about 40 amino acids to about 750 amino acids, about 40 amino acids to about 700 amino acids, about 40 amino acids to about 650 amino acids, about 40 amino acids to about 600 amino acids, about 40 amino acids to about 550 amino acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 450 amino acids, about 40 amino acids to about 400 amino acids, about 40 amino acids to about 350 amino acids, about 40 amino acids to about 300 amino acids, about 40 amino acids to about 280 amino acids, about 40 amino acids to about 260 amino acids, about 40 amino acids to about 240 amino acids, about 40 amino acids to about 220 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 65 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 55 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 1000 amino acids, about 45 amino acids to about 950 amino acids, about 45 amino acids to about 900 amino acids, about 45 amino acids to about 850 amino acids, about 45 amino acids to about 800 amino acids, about 45 amino acids to about 750 amino acids, about 45 amino acids to about 700 amino acids, about 45 amino acids to about 650 amino acids, about 45 amino acids to about 600 amino acids, about 45 amino acids to about 550 amino acids, about 45 amino acids to about 500 amino acids, about 45 amino acids to about 450 amino acids, about 45 amino acids to about 400 amino acids, about 45 amino acids to about 350 amino acids, about 45 amino acids to about 300 amino acids, about 45 amino acids to about 280 amino acids, about 45 amino acids to about 260 amino acids, about 45 amino acids to about 240 amino acids, about 45 amino acids to about 220 amino acids, about 45 amino acids to about 200 amino acids, about 45 amino acids to about 195 amino acids, about 45 amino acids to about 190 amino acids, about 45 amino acids to about 185 amino acids, about 45 amino acids to about 180 amino acids, about 45 amino acids to about 175 amino acids, about 45 amino acids to about 170 amino acids, about 45 amino acids to about 165 amino acids, about 45 amino acids to about 160 amino acids, about 45 amino acids to about 155 amino acids, about 45 amino acids to about 150 amino acids, about 45 amino acids to about 145 amino acids, about 45 amino acids to about 140 amino acids, about 45 amino acids to about 135 amino acids, about 45 amino acids to about 130 amino acids, about 45 amino acids to about 125 amino acids, about 45 amino acids to about 120 amino acids, about 45 amino acids to about 115 amino acids, about 45 amino acids to about 110 amino acids, about 45 amino acids to about 105 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 95 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 85 amino acids, about 45 amino acids to about 80 amino acids, about 45 amino acids to about 75 amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to about 65 amino acids, about 45 amino acids to about 60 amino acids, about 45 amino acids to about 55 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 1000 amino acids, about 50 amino acids to about 950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to about 850 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 145 amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 75 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 65 amino acids, about 50 amino acids to about 60 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 1000 amino acids, about 55 amino acids to about 950 amino acids, about 55 amino acids to about 900 amino acids, about 55 amino acids to about 850 amino acids, about 55 amino acids to about 800 amino acids, about 55 amino acids to about 750 amino acids, about 55 amino acids to about 700 amino acids, about 55 amino acids to about 650 amino acids, about 55 amino acids to about 600 amino acids, about 55 amino acids to about 550 amino acids, about 55 amino acids to about 500 amino acids, about 55 amino acids to about 450 amino acids, about 55 amino acids to about 400 amino acids, about 55 amino acids to about 350 amino acids, about 55 amino acids to about 300 amino acids, about 55 amino acids to about 280 amino acids, about 55 amino acids to about 260 amino acids, about 55 amino acids to about 240 amino acids, about 55 amino acids to about 220 amino acids, about 55 amino acids to about 200 amino acids, about 55 amino acids to about 195 amino acids, about 55 amino acids to about 190 amino acids, about 55 amino acids to about 185 amino acids, about 55 amino acids to about 180 amino acids, about 55 amino acids to about 175 amino acids, about 55 amino acids to about 170 amino acids, about 55 amino acids to about 165 amino acids, about 55 amino acids to about 160 amino acids, about 55 amino acids to about 155 amino acids, about 55 amino acids to about 150 amino acids, about 55 amino acids to about 145 amino acids, about 55 amino acids to about 140 amino acids, about 55 amino acids to about 135 amino acids, about 55 amino acids to about 130 amino acids, about 55 amino acids to about 125 amino acids, about 55 amino acids to about 120 amino acids, about 55 amino acids to about 115 amino acids, about 55 amino acids to about 110 amino acids, about 55 amino acids to about 105 amino acids, about 55 amino acids to about 100 amino acids, about 55 amino acids to about 95 amino acids, about 55 amino acids to about 90 amino acids, about 55 amino acids to about 85 amino acids, about 55 amino acids to about 80 amino acids, about 55 amino acids to about 75 amino acids, about 55 amino acids to about 70 amino acids, about 55 amino acids to about 65 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 1000 amino acids, about 60 amino acids to about 950 amino acids, about 60 amino acids to about 900 amino acids, about 60 amino acids to about 850 amino acids, about 60 amino acids to about 800 amino acids, about 60 amino acids to about 750 amino acids, about 60 amino acids to about 700 amino acids, about 60 amino acids to about 650 amino acids, about 60 amino acids to about 600 amino acids, about 60 amino acids to about 550 amino acids, about 60 amino acids to about 500 amino acids, about 60 amino acids to about 450 amino acids, about 60 amino acids to about 400 amino acids, about 60 amino acids to about 350 amino acids, about 60 amino acids to about 300 amino acids, about 60 amino acids to about 280 amino acids, about 60 amino acids to about 260 amino acids, about 60 amino acids to about 240 amino acids, about 60 amino acids to about 220 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 195 amino acids, about 60 amino acids to about 190 amino acids, about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids, about 60 amino acids to about 65 amino acids, about 65 amino acids to about 1000 amino acids, about 65 amino acids to about 950 amino acids, about 65 amino acids to about 900 amino acids, about 65 amino acids to about 850 amino acids, about 65 amino acids to about 800 amino acids, about 65 amino acids to about 750 amino acids, about 65 amino acids to about 700 amino acids, about 65 amino acids to about 650 amino acids, about 65 amino acids to about 600 amino acids, about 65 amino acids to about 550 amino acids, about 65 amino acids to about 500 amino acids, about 65 amino acids to about 450 amino acids, about 65 amino acids to about 400 amino acids, about 65 amino acids to about 350 amino acids, about 65 amino acids to about 300 amino acids, about 65 amino acids to about 280 amino acids, about 65 amino acids to about 260 amino acids, about 65 amino acids to about 240 amino acids, about 65 amino acids to about 220 amino acids, about 65 amino acids to about 200 amino acids, about 65 amino acids to about 195 amino acids, about 65 amino acids to about 190 amino acids, about 65 amino acids to about 185 amino acids, about 65 amino acids to about 180 amino acids, about 65 amino acids to about 175 amino acids, about 65 amino acids to about 170 amino acids, about 65 amino acids to about 165 amino acids, about 65 amino acids to about 160 amino acids, about 65 amino acids to about 155 amino acids, about 65 amino acids to about 150 amino acids, about 65 amino acids to about 145 amino acids, about 65 amino acids to about 140 amino acids, about 65 amino acids to about 135 amino acids, about 65 amino acids to about 130 amino acids, about 65 amino acids to about 125 amino acids, about 65 amino acids to about 120 amino acids, about 65 amino acids to about 115 amino acids, about 65 amino acids to about 110 amino acids, about 65 amino acids to about 105 amino acids, about 65 amino acids to about 100 amino acids, about 65 amino acids to about 95 amino acids, about 65 amino acids to about 90 amino acids, about 65 amino acids to about 85 amino acids, about 65 amino acids to about 80 amino acids, about 65 amino acids to about 75 amino acids, about 65 amino acids to about 70 amino acids, about 70 amino acids to about 1000 amino acids, about 70 amino acids to about 950 amino acids, about 70 amino acids to about 900 amino acids, about 70 amino acids to about 850 amino acids, about 70 amino acids to about 800 amino acids, about 70 amino acids to about 750 amino acids, about 70 amino acids to about 700 amino acids, about 70 amino acids to about 650 amino acids, about 70 amino acids to about 600 amino acids, about 70 amino acids to about 550 amino acids, about 70 amino acids to about 500 amino acids, about 70 amino acids to about 450 amino acids, about 70 amino acids to about 400 amino acids, about 70 amino acids to about 350 amino acids, about 70 amino acids to about 300 amino acids, about 70 amino acids to about 280 amino acids, about 70 amino acids to about 260 amino acids, about 70 amino acids to about 240 amino acids, about 70 amino acids to about 220 amino acids, about 70 amino acids to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 70 amino acids to about 75 amino acids, about 75 amino acids to about 1000 amino acids, about 75 amino acids to about 950 amino acids, about 75 amino acids to about 900 amino acids, about 75 amino acids to about 850 amino acids, about 75 amino acids to about 800 amino acids, about 75 amino acids to about 750 amino acids, about 75 amino acids to about 700 amino acids, about 75 amino acids to about 650 amino acids, about 75 amino acids to about 600 amino acids, about 75 amino acids to about 550 amino acids, about 75 amino acids to about 500 amino acids, about 75 amino acids to about 450 amino acids, about 75 amino acids to about 400 amino acids, about 75 amino acids to about 350 amino acids, about 75 amino acids to about 300 amino acids, about 75 amino acids to about 280 amino acids, about 75 amino acids to about 260 amino acids, about 75 amino acids to about 240 amino acids, about 75 amino acids to about 220 amino acids, about 75 amino acids to about 200 amino acids, about 75 amino acids to about 195 amino acids, about 75 amino acids to about 190 amino acids, about 75 amino acids to about 185 amino acids, about 75 amino acids to about 180 amino acids, about 75 amino acids to about 175 amino acids, about 75 amino acids to about 170 amino acids, about 75 amino acids to about 165 amino acids, about 75 amino acids to about 160 amino acids, about 75 amino acids to about 155 amino acids, about 75 amino acids to about 150 amino acids, about 75 amino acids to about 145 amino acids, about 75 amino acids to about 140 amino acids, about 75 amino acids to about 135 amino acids, about 75 amino acids to about 130 amino acids, about 75 amino acids to about 125 amino acids, about 75 amino acids to about 120 amino acids, about 75 amino acids to about 115 amino acids, about 75 amino acids to about 110 amino acids, about 75 amino acids to about 105 amino acids, about 75 amino acids to about 100 amino acids, about 75 amino acids to about 95 amino acids, about 75 amino acids to about 90 amino acids, about 75 amino acids to about 85 amino acids, about 75 amino acids to about 80 amino acids, about 80 amino acids to about 1000 amino acids, about 80 amino acids to about 950 amino acids, about 80 amino acids to about 900 amino acids, about 80 amino acids to about 850 amino acids, about 80 amino acids to about 800 amino acids, about 80 amino acids to about 750 amino acids, about 80 amino acids to about 700 amino acids, about 80 amino acids to about 650 amino acids, about 80 amino acids to about 600 amino acids, about 80 amino acids to about 550 amino acids, about 80 amino acids to about 500 amino acids, about 80 amino acids to about 450 amino acids, about 80 amino acids to about 400 amino acids, about 80 amino acids to about 350 amino acids, about 80 amino acids to about 300 amino acids, about 80 amino acids to about 280 amino acids, about 80 amino acids to about 260 amino acids, about 80 amino acids to about 240 amino acids, about 80 amino acids to about 220 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 175 amino acids, about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 80 amino acids to about 85 amino acids, about 85 amino acids to about 1000 amino acids, about 85 amino acids to about 950 amino acids, about 85 amino acids to about 900 amino acids, about 85 amino acids to about 850 amino acids, about 85 amino acids to about 800 amino acids, about 85 amino acids to about 750 amino acids, about 85 amino acids to about 700 amino acids, about 85 amino acids to about 650 amino acids, about 85 amino acids to about 600 amino acids, about 85 amino acids to about 550 amino acids, about 85 amino acids to about 500 amino acids, about 85 amino acids to about 450 amino acids, about 85 amino acids to about 400 amino acids, about 85 amino acids to about 350 amino acids, about 85 amino acids to about 300 amino acids, about 85 amino acids to about 280 amino acids, about 85 amino acids to about 260 amino acids, about 85 amino acids to about 240 amino acids, about 85 amino acids to about 220 amino acids, about 85 amino acids to about 200 amino acids, about 85 amino acids to about 195 amino acids, about 85 amino acids to about 190 amino acids, about 85 amino acids to about 185 amino acids, about 85 amino acids to about 180 amino acids, about 85 amino acids to about 175 amino acids, about 85 amino acids to about 170 amino acids, about 85 amino acids to about 165 amino acids, about 85 amino acids to about 160 amino acids, about 85 amino acids to about 155 amino acids, about 85 amino acids to about 150 amino acids, about 85 amino acids to about 145 amino acids, about 85 amino acids to about 140 amino acids, about 85 amino acids to about 135 amino acids, about 85 amino acids to about 130 amino acids, about 85 amino acids to about 125 amino acids, about 85 amino acids to about 120 amino acids, about 85 amino acids to about 115 amino acids, about 85 amino acids to about 110 amino acids, about 85 amino acids to about 105 amino acids, about 85 amino acids to about 100 amino acids, about 85 amino acids to about 95 amino acids, about 85 amino acids to about 90 amino acids, about 90 amino acids to about 1000 amino acids, about 90 amino acids to about 950 amino acids, about 90 amino acids to about 900 amino acids, about 90 amino acids to about 850 amino acids, about 90 amino acids to about 800 amino acids, about 90 amino acids to about 750 amino acids, about 90 amino acids to about 700 amino acids, about 90 amino acids to about 650 amino acids, about 90 amino acids to about 600 amino acids, about 90 amino acids to about 550 amino acids, about 90 amino acids to about 500 amino acids, about 90 amino acids to about 450 amino acids, about 90 amino acids to about 400 amino acids, about 90 amino acids to about 350 amino acids, about 90 amino acids to about 300 amino acids, about 90 amino acids to about 280 amino acids, about 90 amino acids to about 260 amino acids, about 90 amino acids to about 240 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 90 amino acids to about 95 amino acids, about 95 amino acids to about 1000 amino acids, about 95 amino acids to about 950 amino acids, about 95 amino acids to about 900 amino acids, about 95 amino acids to about 850 amino acids, about 95 amino acids to about 800 amino acids, about 95 amino acids to about 750 amino acids, about 95 amino acids to about 700 amino acids, about 95 amino acids to about 650 amino acids, about 95 amino acids to about 600 amino acids, about 95 amino acids to about 550 amino acids, about 95 amino acids to about 500 amino acids, about 95 amino acids to about 450 amino acids, about 95 amino acids to about 400 amino acids, about 95 amino acids to about 350 amino acids, about 95 amino acids to about 300 amino acids, about 95 amino acids to about 280 amino acids, about 95 amino acids to about 260 amino acids, about 95 amino acids to about 240 amino acids, about 95 amino acids to about 220 amino acids, about 95 amino acids to about 200 amino acids, about 95 amino acids to about 195 amino acids, about 95 amino acids to about 190 amino acids, about 95 amino acids to about 185 amino acids, about 95 amino acids to about 180 amino acids, about 95 amino acids to about 175 amino acids, about 95 amino acids to about 170 amino acids, about 95 amino acids to about 165 amino acids, about 95 amino acids to about 160 amino acids, about 95 amino acids to about 155 amino acids, about 95 amino acids to about 150 amino acids, about 95 amino acids to about 145 amino acids, about 95 amino acids to about 140 amino acids, about 95 amino acids to about 135 amino acids, about 95 amino acids to about 130 amino acids, about 95 amino acids to about 125 amino acids, about 95 amino acids to about 120 amino acids, about 95 amino acids to about 115 amino acids, about 95 amino acids to about 110 amino acids, about 95 amino acids to about 105 amino acids, about 95 amino acids to about 100 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 280 amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 100 amino acids to about 105 amino acids, about 105 amino acids to about 1000 amino acids, about 105 amino acids to about 950 amino acids, about 105 amino acids to about 900 amino acids, about 105 amino acids to about 850 amino acids, about 105 amino acids to about 800 amino acids, about 105 amino acids to about 750 amino acids, about 105 amino acids to about 700 amino acids, about 105 amino acids to about 650 amino acids, about 105 amino acids to about 600 amino acids, about 105 amino acids to about 550 amino acids, about 105 amino acids to about 500 amino acids, about 105 amino acids to about 450 amino acids, about 105 amino acids to about 400 amino acids, about 105 amino acids to about 350 amino acids, about 105 amino acids to about 300 amino acids, about 105 amino acids to about 280 amino acids, about 105 amino acids to about 260 amino acids, about 105 amino acids to about 240 amino acids, about 105 amino acids to about 220 amino acids, about 105 amino acids to about 200 amino acids, about 105 amino acids to about 195 amino acids, about 105 amino acids to about 190 amino acids, about 105 amino acids to about 185 amino acids, about 105 amino acids to about 180 amino acids, about 105 amino acids to about 175 amino acids, about 105 amino acids to about 170 amino acids, about 105 amino acids to about 165 amino acids, about 105 amino acids to about 160 amino acids, about 105 amino acids to about 155 amino acids, about 105 amino acids to about 150 amino acids, about 105 amino acids to about 145 amino acids, about 105 amino acids to about 140 amino acids, about 105 amino acids to about 135 amino acids, about 105 amino acids to about 130 amino acids, about 105 amino acids to about 125 amino acids, about 105 amino acids to about 120 amino acids, about 105 amino acids to about 115 amino acids, about 105 amino acids to about 110 amino acids, about 110 amino acids to about 1000 amino acids, about 110 amino acids to about 950 amino acids, about 110 amino acids to about 900 amino acids, about 110 amino acids to about 850 amino acids, about 110 amino acids to about 800 amino acids, about 110 amino acids to about 750 amino acids, about 110 amino acids to about 700 amino acids, about 110 amino acids to about 650 amino acids, about 110 amino acids to about 600 amino acids, about 110 amino acids to about 550 amino acids, about 110 amino acids to about 500 amino acids, about 110 amino acids to about 450 amino acids, about 110 amino acids to about 400 amino acids, about 110 amino acids to about 350 amino acids, about 110 amino acids to about 300 amino acids, about 110 amino acids to about 280 amino acids, about 110 amino acids to about 260 amino acids, about 110 amino acids to about 240 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 1000 amino acids, about 115 amino acids to about 950 amino acids, about 115 amino acids to about 900 amino acids, about 115 amino acids to about 850 amino acids, about 115 amino acids to about 800 amino acids, about 115 amino acids to about 750 amino acids, about 115 amino acids to about 700 amino acids, about 115 amino acids to about 650 amino acids, about 115 amino acids to about 600 amino acids, about 115 amino acids to about 550 amino acids, about 115 amino acids to about 500 amino acids, about 115 amino acids to about 450 amino acids, about 115 amino acids to about 400 amino acids, about 115 amino acids to about 350 amino acids, about 115 amino acids to about 300 amino acids, about 115 amino acids to about 280 amino acids, about 115 amino acids to about 260 amino acids, about 115 amino acids to about 240 amino acids, about 115 amino acids to about 220 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 1000 amino acids, about 120 amino acids to about 950 amino acids, about 120 amino acids to about 900 amino acids, about 120 amino acids to about 850 amino acids, about 120 amino acids to about 800 amino acids, about 120 amino acids to about 750 amino acids, about 120 amino acids to about 700 amino acids, about 120 amino acids to about 650 amino acids, about 120 amino acids to about 600 amino acids, about 120 amino acids to about 550 amino acids, about 120 amino acids to about 500 amino acids, about 120 amino acids to about 450 amino acids, about 120 amino acids to about 400 amino acids, about 120 amino acids to about 350 amino acids, about 120 amino acids to about 300 amino acids, about 120 amino acids to about 280 amino acids, about 120 amino acids to about 260 amino acids, about 120 amino acids to about 240 amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to about 185 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 175 amino acids, about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to about 150 amino acids, about 120 amino acids to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 1000 amino acids, about 125 amino acids to about 950 amino acids, about 125 amino acids to about 900 amino acids, about 125 amino acids to about 850 amino acids, about 125 amino acids to about 800 amino acids, about 125 amino acids to about 750 amino acids, about 125 amino acids to about 700 amino acids, about 125 amino acids to about 650 amino acids, about 125 amino acids to about 600 amino acids, about 125 amino acids to about 550 amino acids, about 125 amino acids to about 500 amino acids, about 125 amino acids to about 450 amino acids, about 125 amino acids to about 400 amino acids, about 125 amino acids to about 350 amino acids, about 125 amino acids to about 300 amino acids, about 125 amino acids to about 280 amino acids, about 125 amino acids to about 260 amino acids, about 125 amino acids to about 240 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids, about 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 1000 amino acids, about 130 amino acids to about 950 amino acids, about 130 amino acids to about 900 amino acids, about 130 amino acids to about 850 amino acids, about 130 amino acids to about 800 amino acids, about 130 amino acids to about 750 amino acids, about 130 amino acids to about 700 amino acids, about 130 amino acids to about 650 amino acids, about 130 amino acids to about 600 amino acids, about 130 amino acids to about 550 amino acids, about 130 amino acids to about 500 amino acids, about 130 amino acids to about 450 amino acids, about 130 amino acids to about 400 amino acids, about 130 amino acids to about 350 amino acids, about 130 amino acids to about 300 amino acids, about 130 amino acids to about 280 amino acids, about 130 amino acids to about 260 amino acids, about 130 amino acids to about 240 amino acids, about 130 amino acids to about 220 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 1000 amino acids, about 135 amino acids to about 950 amino acids, about 135 amino acids to about 900 amino acids, about 135 amino acids to about 850 amino acids, about 135 amino acids to about 800 amino acids, about 135 amino acids to about 750 amino acids, about 135 amino acids to about 700 amino acids, about 135 amino acids to about 650 amino acids, about 135 amino acids to about 600 amino acids, about 135 amino acids to about 550 amino acids, about 135 amino acids to about 500 amino acids, about 135 amino acids to about 450 amino acids, about 135 amino acids to about 400 amino acids, about 135 amino acids to about 350 amino acids, about 135 amino acids to about 300 amino acids, about 135 amino acids to about 280 amino acids, about 135 amino acids to about 260 amino acids, about 135 amino acids to about 240 amino acids, about 135 amino acids to about 220 amino acids, about 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids, about 135 amino acids to about 140 amino acids, about 140 amino acids to about 1000 amino acids, about 140 amino acids to about 950 amino acids, about 140 amino acids to about 900 amino acids, about 140 amino acids to about 850 amino acids, about 140 amino acids to about 800 amino acids, about 140 amino acids to about 750 amino acids, about 140 amino acids to about 700 amino acids, about 140 amino acids to about 650 amino acids, about 140 amino acids to about 600 amino acids, about 140 amino acids to about 550 amino acids, about 140 amino acids to about 500 amino acids, about 140 amino acids to about 450 amino acids, about 140 amino acids to about 400 amino acids, about 140 amino acids to about 350 amino acids, about 140 amino acids to about 300 amino acids, about 140 amino acids to about 280 amino acids, about 140 amino acids to about 260 amino acids, about 140 amino acids to about 240 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140 amino acids to about 145 amino acids, about 145 amino acids to about 1000 amino acids, about 145 amino acids to about 950 amino acids, about 145 amino acids to about 900 amino acids, about 145 amino acids to about 850 amino acids, about 145 amino acids to about 800 amino acids, about 145 amino acids to about 750 amino acids, about 145 amino acids to about 700 amino acids, about 145 amino acids to about 650 amino acids, about 145 amino acids to about 600 amino acids, about 145 amino acids to about 550 amino acids, about 145 amino acids to about 500 amino acids, about 145 amino acids to about 450 amino acids, about 145 amino acids to about 400 amino acids, about 145 amino acids to about 350 amino acids, about 145 amino acids to about 300 amino acids, about 145 amino acids to about 280 amino acids, about 145 amino acids to about 260 amino acids, about 145 amino acids to about 240 amino acids, about 145 amino acids to about 220 amino acids, about 145 amino acids to about 200 amino acids, about 145 amino acids to about 195 amino acids, about 145 amino acids to about 190 amino acids, about 145 amino acids to about 185 amino acids, about 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 1000 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids to about 850 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 280 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 1000 amino acids, about 155 amino acids to about 950 amino acids, about 155 amino acids to about 900 amino acids, about 155 amino acids to about 850 amino acids, about 155 amino acids to about 800 amino acids, about 155 amino acids to about 750 amino acids, about 155 amino acids to about 700 amino acids, about 155 amino acids to about 650 amino acids, about 155 amino acids to about 600 amino acids, about 155 amino acids to about 550 amino acids, about 155 amino acids to about 500 amino acids, about 155 amino acids to about 450 amino acids, about 155 amino acids to about 400 amino acids, about 155 amino acids to about 350 amino acids, about 155 amino acids to about 300 amino acids, about 155 amino acids to about 280 amino acids, about 155 amino acids to about 260 amino acids, about 155 amino acids to about 240 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 1000 amino acids, about 160 amino acids to about 950 amino acids, about 160 amino acids to about 900 amino acids, about 160 amino acids to about 850 amino acids, about 160 amino acids to about 800 amino acids, about 160 amino acids to about 750 amino acids, about 160 amino acids to about 700 amino acids, about 160 amino acids to about 650 amino acids, about 160 amino acids to about 600 amino acids, about 160 amino acids to about 550 amino acids, about 160 amino acids to about 500 amino acids, about 160 amino acids to about 450 amino acids, about 160 amino acids to about 400 amino acids, about 160 amino acids to about 350 amino acids, about 160 amino acids to about 300 amino acids, about 160 amino acids to about 280 amino acids, about 160 amino acids to about 260 amino acids, about 160 amino acids to about 240 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 amino acids to about 190 amino acids, about 160 amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids, about 165 amino acids to about 1000 amino acids, about 165 amino acids to about 950 amino acids, about 165 amino acids to about 900 amino acids, about 165 amino acids to about 850 amino acids, about 165 amino acids to about 800 amino acids, about 165 amino acids to about 750 amino acids, about 165 amino acids to about 700 amino acids, about 165 amino acids to about 650 amino acids, about 165 amino acids to about 600 amino acids, about 165 amino acids to about 550 amino acids, about 165 amino acids to about 500 amino acids, about 165 amino acids to about 450 amino acids, about 165 amino acids to about 400 amino acids, about 165 amino acids to about 350 amino acids, about 165 amino acids to about 300 amino acids, about 165 amino acids to about 280 amino acids, about 165 amino acids to about 260 amino acids, about 165 amino acids to about 240 amino acids, about 165 amino acids to about 220 amino acids, about 165 amino acids to about 200 amino acids, about 165 amino acids to about 195 amino acids, about 165 amino acids to about 190 amino acids, about 165 amino acids to about 185 amino acids, about 165 amino acids to about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 1000 amino acids, about 170 amino acids to about 950 amino acids, about 170 amino acids to about 900 amino acids, about 170 amino acids to about 850 amino acids, about 170 amino acids to about 800 amino acids, about 170 amino acids to about 750 amino acids, about 170 amino acids to about 700 amino acids, about 170 amino acids to about 650 amino acids, about 170 amino acids to about 600 amino acids, about 170 amino acids to about 550 amino acids, about 170 amino acids to about 500 amino acids, about 170 amino acids to about 450 amino acids, about 170 amino acids to about 400 amino acids, about 170 amino acids to about 350 amino acids, about 170 amino acids to about 300 amino acids, about 170 amino acids to about 280 amino acids, about 170 amino acids to about 260 amino acids, about 170 amino acids to about 240 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 1000 amino acids, about 175 amino acids to about 950 amino acids, about 175 amino acids to about 900 amino acids, about 175 amino acids to about 850 amino acids, about 175 amino acids to about 800 amino acids, about 175 amino acids to about 750 amino acids, about 175 amino acids to about 700 amino acids, about 175 amino acids to about 650 amino acids, about 175 amino acids to about 600 amino acids, about 175 amino acids to about 550 amino acids, about 175 amino acids to about 500 amino acids, about 175 amino acids to about 450 amino acids, about 175 amino acids to about 400 amino acids, about 175 amino acids to about 350 amino acids, about 175 amino acids to about 300 amino acids, about 175 amino acids to about 280 amino acids, about 175 amino acids to about 260 amino acids, about 175 amino acids to about 240 amino acids, about 175 amino acids to about 220 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to about 190 amino acids, about 175 amino acids to about 185 amino acids, about 175 amino acids to about 180 amino acids, about 180 amino acids to about 1000 amino acids, about 180 amino acids to about 950 amino acids, about 180 amino acids to about 900 amino acids, about 180 amino acids to about 850 amino acids, about 180 amino acids to about 800 amino acids, about 180 amino acids to about 750 amino acids, about 180 amino acids to about 700 amino acids, about 180 amino acids to about 650 amino acids, about 180 amino acids to about 600 amino acids, about 180 amino acids to about 550 amino acids, about 180 amino acids to about 500 amino acids, about 180 amino acids to about 450 amino acids, about 180 amino acids to about 400 amino acids, about 180 amino acids to about 350 amino acids, about 180 amino acids to about 300 amino acids, about 180 amino acids to about 280 amino acids, about 180 amino acids to about 260 amino acids, about 180 amino acids to about 240 amino acids, about 180 amino acids to about 220 amino acids, about 180 amino acids to about 200 amino acids, about 180 amino acids to about 195 amino acids, about 180 amino acids to about 190 amino acids, about 180 amino acids to about 185 amino acids, about 185 amino acids to about 1000 amino acids, about 185 amino acids to about 950 amino acids, about 185 amino acids to about 900 amino acids, about 185 amino acids to about 850 amino acids, about 185 amino acids to about 800 amino acids, about 185 amino acids to about 750 amino acids, about 185 amino acids to about 700 amino acids, about 185 amino acids to about 650 amino acids, about 185 amino acids to about 600 amino acids, about 185 amino acids to about 550 amino acids, about 185 amino acids to about 500 amino acids, about 185 amino acids to about 450 amino acids, about 185 amino acids to about 400 amino acids, about 185 amino acids to about 350 amino acids, about 185 amino acids to about 300 amino acids, about 185 amino acids to about 280 amino acids, about 185 amino acids to about 260 amino acids, about 185 amino acids to about 240 amino acids, about 185 amino acids to about 220 amino acids, about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195 amino acids, about 185 amino acids to about 190 amino acids, about 190 amino acids to about 1000 amino acids, about 190 amino acids to about 950 amino acids, about 190 amino acids to about 900 amino acids, about 190 amino acids to about 850 amino acids, about 190 amino acids to about 800 amino acids, about 190 amino acids to about 750 amino acids, about 190 amino acids to about 700 amino acids, about 190 amino acids to about 650 amino acids, about 190 amino acids to about 600 amino acids, about 190 amino acids to about 550 amino acids, about 190 amino acids to about 500 amino acids, about 190 amino acids to about 450 amino acids, about 190 amino acids to about 400 amino acids, about 190 amino acids to about 350 amino acids, about 190 amino acids to about 300 amino acids, about 190 amino acids to about 280 amino acids, about 190 amino acids to about 260 amino acids, about 190 amino acids to about 240 amino acids, about 190 amino acids to about 220 amino acids, about 190 amino acids to about 200 amino acids, about 190 amino acids to about 195 amino acids, about 195 amino acids to about 1000 amino acids, about 195 amino acids to about 950 amino acids, about 195 amino acids to about 900 amino acids, about 195 amino acids to about 850 amino acids, about 195 amino acids to about 800 amino acids, about 195 amino acids to about 750 amino acids, about 195 amino acids to about 700 amino acids, about 195 amino acids to about 650 amino acids, about 195 amino acids to about 600 amino acids, about 195 amino acids to about 550 amino acids, about 195 amino acids to about 500 amino acids, about 195 amino acids to about 450 amino acids, about 195 amino acids to about 400 amino acids, about 195 amino acids to about 350 amino acids, about 195 amino acids to about 300 amino acids, about 195 amino acids to about 280 amino acids, about 195 amino acids to about 260 amino acids, about 195 amino acids to about 240 amino acids, about 195 amino acids to about 220 amino acids, about 195 amino acids to about 200 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 amino acids, about 220 amino acids to about 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 500 amino acids, about 220 amino acids to about 450 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 350 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids, about 240 amino acids to about 450 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 350 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 450 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 350 amino acids, about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 450 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 350 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 350 amino acids, about 350 amino acids to about 1000 amino acids, about 350 amino acids to about 950 amino acids, about 350 amino acids to about 900 amino acids, about 350 amino acids to about 850 amino acids, about 350 amino acids to about 800 amino acids, about 350 amino acids to about 750 amino acids, about 350 amino acids to about 700 amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids to about 600 amino acids, about 350 amino acids to about 550 amino acids, about 350 amino acids to about 500 amino acids, about 350 amino acids to about 450 amino acids, about 350 amino acids to about 400 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 450 amino acids, about 450 amino acids to about 1000 amino acids, about 450 amino acids to about 950 amino acids, about 450 amino acids to about 900 amino acids, about 450 amino acids to about 850 amino acids, about 450 amino acids to about 800 amino acids, about 450 amino acids to about 750 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550 amino acids, about 450 amino acids to about 500 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids, about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 1000 amino acids, about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids, or about 950 amino acids to about 1000 amino acids.

Any of the target-binding domains described herein can bind to its target with a dissociation equilibrium constant (KD) of less than 1 x 10 ⁷ M, less than 1 x 10 ⁸ M, less than 1 x 10 ⁹ M, less than 1 x 10 ¹⁰ M, less than 1 x 10 ¹¹ M, less than 1 x 10 ¹² M, or less than 1 x 10 ¹³ M. In some embodiments, the antigen-binding protein constructs provided herein can bind to an identifying antigen with a KD of about 1 x 10 ³ M to about 1 x 10 ⁵ M, about 1 x 10 ⁴ M to about 1 x 10 ⁶ M, about 1 x 10 ⁵ M to about 1 x 10 ⁷ M, about 1 x 10 ⁶ M to about 1 x 10 ⁸ M, about 1 x 10 ⁷ M to about 1 x 10 ⁹ M, about 1 x 10 ⁸ M to about 1 x 10 ¹⁰ M, or about 1 x 10 ⁹ M to about 1 x l0 ^u M (inclusive).

Any of the target-binding domains described herein can bind to its target with a KD of between about 1 pM to about 30 nM (e.g., about 1 pM to about 25 nM, about 1 pM to about 20 nM, about 1 pM to about 15 nM, about 1 pM to about 10 nM, about 1 pM to about 5 nM, about 1 pM to about 2 nM, about 1 pM to about 1 nM, about 1 pM to about

950 pM, about 1 pM to about 900 pM, about 1 pM to about 850 pM, about 1 pM to about

800 pM, about 1 pM to about 750 pM, about 1 pM to about 700 pM, about 1 pM to about

650 pM, about 1 pM to about 600 pM, about 1 pM to about 550 pM, about 1 pM to about

500 pM, about 1 pM to about 450 pM, about 1 pM to about 400 pM, about 1 pM to about 350 pM, about 1 pM to about 300 pM, about 1 pM to about 250 pM, about 1 pM to about

200 pM, about 1 pM to about 150 pM, about 1 pM to about 100 pM, about 1 pM to about 90 pM, about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM to about 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 4 pM, about 1 pM to about 3 pM, about 1 pM to about 2 pM, about 2 pM to about 30 nM, about 2 pM to about 25 nM, about 2 pM to about 20 nM, about 2 pM to about 15 nM, about 2 pM to about 10 nM, about 2 pM to about 5 nM, about 2 pM to about 2 nM, about 2 pM to about 1 nM, about 2 pM to about 950 pM, about 2 pM to about 900 pM, about 2 pM to about 850 pM, about 2 pM to about 800 pM, about 2 pM to about 750 pM, about 2 pM to about 700 pM, about 2 pM to about 650 pM, about 2 pM to about 600 pM, about 2 pM to about 550 pM, about 2 pM to about 500 pM, about 2 pM to about 450 pM, about 2 pM to about 400 pM, about 2 pM to about 350 pM, about 2 pM to about 300 pM, about 2 pM to about 250 pM, about 2 pM to about 200 pM, about 2 pM to about 150 pM, about 2 pM to about 100 pM, about 2 pM to about 90 pM, about 2 pM to about 80 pM, about 2 pM to about 70 pM, about 2 pM to about 60 pM, about 2 pM to about 50 pM, about 2 pM to about 40 pM, about 2 pM to about 30 pM, about 2 pM to about 20 pM, about 2 pM to about 10 pM, about 2 pM to about 5 pM, about 2 pM to about 4 pM, about 2 pM to about 3 pM, about 5 pM to about 30 nM, about 5 pM to about 25 nM, about 5 pM to about 20 nM, about 5 pM to about 15 nM, about 5 pM to about 10 nM, about 5 pM to about 5 nM, about 5 pM to about 2 nM, about 5 pM to about 1 nM, about 5 pM to about 950 pM, about 5 pM to about 900 pM, about 5 pM to about 850 pM, about 5 pM to about 800 pM, about 5 pM to about 750 pM, about 5 pM to about 700 pM, about 5 pM to about 650 pM, about 5 pM to about 600 pM, about 5 pM to about 550 pM, about 5 pM to about 500 pM, about 5 pM to about 450 pM, about 5 pM to about 400 pM, about 5 pM to about 350 pM, about 5 pM to about 300 pM, about 5 pM to about 250 pM, about 5 pM to about 200 pM, about 5 pM to about 150 pM, about 5 pM to about 100 pM, about 5 pM to about 90 pM, about 5 pM to about 80 pM, about 5 pM to about 70 pM, about 5 pM to about 60 pM, about 5 pM to about 50 pM, about 5 pM to about 40 pM, about 5 pM to about 30 pM, about 5 pM to about 20 pM, about 5 pM to about 10 pM, about 10 pM to about 30 nM, about 10 pM to about 25 nM, about 10 pM to about 20 nM, about 10 pM to about 15 nM, about 10 pM to about 10 nM, about 10 pM to about 5 nM, about 10 pM to about 2 nM, about 10 pM to about 1 nM, about 10 pM to about 950 pM, about 10 pM to about 900 pM, about 10 pM to about 850 pM, about 10 pM to about 800 pM, about 10 pM to about 750 pM, about 10 pM to about 700 pM, about 10 pM to about 650 pM, about 10 pM to about 600 pM, about 10 pM to about 550 pM, about 10 pM to about 500 pM, about 10 pM to about 450 pM, about 10 pM to about 400 pM, about 10 pM to about 350 pM, about 10 pM to about 300 pM, about 10 pM to about 250 pM, about 10 pM to about 200 pM, about 10 pM to about 150 pM, about 10 pM to about 100 pM, about 10 pM to about 90 pM, about 10 pM to about 80 pM, about 10 pM to about 70 pM, about 10 pM to about 60 pM, about 10 pM to about 50 pM, about 10 pM to about 40 pM, about 10 pM to about 30 pM, about 10 pM to about 20 pM, about 15 pM to about 30 nM, about 15 pM to about 25 nM, about 15 pM to about 20 nM, about 15 pM to about 15 nM, about 15 pM to about 10 nM, about 15 pM to about 5 nM, about 15 pM to about 2 nM, about 15 pM to about 1 nM, about 15 pM to about 950 pM, about 15 pM to about 900 pM, about 15 pM to about 850 pM, about 15 pM to about 800 pM, about 15 pM to about 750 pM, about 15 pM to about 700 pM, about 15 pM to about 650 pM, about 15 pM to about 600 pM, about 15 pM to about 550 pM, about 15 pM to about 500 pM, about 15 pM to about 450 pM, about 15 pM to about 400 pM, about 15 pM to about 350 pM, about 15 pM to about 300 pM, about 15 pM to about 250 pM, about 15 pM to about 200 pM, about 15 pM to about 150 pM, about 15 pM to about 100 pM, about 15 pM to about 90 pM, about 15 pM to about 80 pM, about 15 pM to about 70 pM, about 15 pM to about 60 pM, about 15 pM to about 50 pM, about 15 pM to about 40 pM, about 15 pM to about 30 pM, about 15 pM to about 20 pM, about 20 pM to about 30 nM, about 20 pM to about 25 nM, about 20 pM to about 20 nM, about 20 pM to about 15 nM, about 20 pM to about 10 nM, about 20 pM to about 5 nM, about 20 pM to about 2 nM, about 20 pM to about 1 nM, about 20 pM to about 950 pM, about 20 pM to about 900 pM, about 20 pM to about 850 pM, about 20 pM to about 800 pM, about 20 pM to about 750 pM, about 20 pM to about 700 pM, about 20 pM to about 650 pM, about 20 pM to about 600 pM, about 20 pM to about 550 pM, about 20 pM to about 500 pM, about 20 pM to about 450 pM, about 20 pM to about 400 pM, about 20 pM to about 350 pM, about 20 pM to about 300 pM, about 20 pM to about 250 pM, about 20 pM to about 20 pM, about 200 pM to about 150 pM, about 20 pM to about 100 pM, about 20 pM to about 90 pM, about 20 pM to about 80 pM, about 20 pM to about 70 pM, about 20 pM to about 60 pM, about 20 pM to about 50 pM, about 20 pM to about 40 pM, about 20 pM to about 30 pM, about 30 pM to about 30 nM, about 30 pM to about 25 nM, about 30 pM to about 30 nM, about 30 pM to about 15 nM, about 30 pM to about 10 nM, about 30 pM to about 5 nM, about 30 pM to about 2 nM, about 30 pM to about 1 nM, about 30 pM to about 950 pM, about 30 pM to about 900 pM, about 30 pM to about 850 pM, about 30 pM to about 800 pM, about 30 pM to about 750 pM, about 30 pM to about 700 pM, about 30 pM to about 650 pM, about 30 pM to about 600 pM, about 30 pM to about 550 pM, about 30 pM to about 500 pM, about 30 pM to about 450 pM, about 30 pM to about 400 pM, about 30 pM to about 350 pM, about 30 pM to about 300 pM, about 30 pM to about 250 pM, about 30 pM to about 200 pM, about 30 pM to about 150 pM, about 30 pM to about 100 pM, about 30 pM to about 90 pM, about 30 pM to about 80 pM, about 30 pM to about 70 pM, about 30 pM to about 60 pM, about 30 pM to about 50 pM, about 30 pM to about 40 pM, about 40 pM to about 30 nM, about 40 pM to about 25 nM, about 40 pM to about 30 nM, about 40 pM to about 15 nM, about 40 pM to about 10 nM, about 40 pM to about 5 nM, about 40 pM to about 2 nM, about 40 pM to about 1 nM, about 40 pM to about 950 pM, about 40 pM to about 900 pM, about 40 pM to about 850 pM, about 40 pM to about 800 pM, about 40 pM to about 750 pM, about 40 pM to about 700 pM, about 40 pM to about 650 pM, about 40 pM to about 600 pM, about 40 pM to about 550 pM, about 40 pM to about 500 pM, about 40 pM to about 450 pM, about 40 pM to about 400 pM, about 40 pM to about 350 pM, about 40 pM to about 300 pM, about 40 pM to about 250 pM, about 40 pM to about 200 pM, about 40 pM to about 150 pM, about 40 pM to about 100 pM, about 40 pM to about 90 pM, about 40 pM to about 80 pM, about 40 pM to about 70 pM, about 40 pM to about 60 pM, about 40 pM to about 50 pM, about 50 pM to about 30 nM, about 50 pM to about 25 nM, about 50 pM to about 30 nM, about 50 pM to about 15 nM, about 50 pM to about 10 nM, about 50 pM to about 5 nM, about 50 pM to about 2 nM, about 50 pM to about 1 nM, about 50 pM to about 950 pM, about 50 pM to about 900 pM, about 50 pM to about 850 pM, about 50 pM to about 800 pM, about 50 pM to about 750 pM, about 50 pM to about 700 pM, about 50 pM to about 650 pM, about 50 pM to about 600 pM, about 50 pM to about 550 pM, about 50 pM to about 500 pM, about 50 pM to about 450 pM, about 50 pM to about 400 pM, about 50 pM to about 350 pM, about 50 pM to about 300 pM, about 50 pM to about 250 pM, about 50 pM to about 200 pM, about 50 pM to about 150 pM, about 50 pM to about 100 pM, about 50 pM to about 90 pM, about 50 pM to about 80 pM, about 50 pM to about 70 pM, about 50 pM to about 60 pM, about 60 pM to about 30 nM, about 60 pM to about 25 nM, about 60 pM to about 30 nM, about 60 pM to about 15 nM, about 60 pM to about 10 nM, about 60 pM to about 5 nM, about 60 pM to about 2 nM, about 60 pM to about 1 nM, about 60 pM to about 950 pM, about 60 pM to about 900 pM, about 60 pM to about 850 pM, about 60 pM to about 800 pM, about 60 pM to about 750 pM, about 60 pM to about 700 pM, about 60 pM to about 650 pM, about 60 pM to about 600 pM, about 60 pM to about 550 pM, about 60 pM to about 500 pM, about 60 pM to about 450 pM, about 60 pM to about 400 pM, about 60 pM to about 350 pM, about 60 pM to about 300 pM, about 60 pM to about 250 pM, about 60 pM to about 200 pM, about 60 pM to about 150 pM, about 60 pM to about 100 pM, about 60 pM to about 90 pM, about 60 pM to about 80 pM, about 60 pM to about 70 pM, about 70 pM to about 30 nM, about 70 pM to about 25 nM, about 70 pM to about 30 nM, about 70 pM to about 15 nM, about 70 pM to about 10 nM, about 70 pM to about 5 nM, about 70 pM to about 2 nM, about 70 pM to about 1 nM, about 70 pM to about 950 pM, about 70 pM to about 900 pM, about 70 pM to about 850 pM, about 70 pM to about 800 pM, about 70 pM to about 750 pM, about 70 pM to about 700 pM, about 70 pM to about 650 pM, about 70 pM to about 600 pM, about 70 pM to about 550 pM, about 70 pM to about 500 pM, about 70 pM to about 450 pM, about 70 pM to about 400 pM, about 70 pM to about 350 pM, about 70 pM to about 300 pM, about 70 pM to about 250 pM, about 70 pM to about 200 pM, about 70 pM to about 150 pM, about 70 pM to about 100 pM, about 70 pM to about 90 pM, about 70 pM to about 80 pM, about 80 pM to about 30 nM, about 80 pM to about 25 nM, about 80 pM to about 30 nM, about 80 pM to about 15 nM, about 80 pM to about 10 nM, about 80 pM to about 5 nM, about 80 pM to about 2 nM, about 80 pM to about 1 nM, about 80 pM to about 950 pM, about 80 pM to about 900 pM, about 80 pM to about

850 pM, about 80 pM to about 800 pM, about 80 pM to about 750 pM, about 80 pM to about 700 pM, about 80 pM to about 650 pM, about 80 pM to about 600 pM, about 80 pM to about 550 pM, about 80 pM to about 500 pM, about 80 pM to about 450 pM, about 80 pM to about 400 pM, about 80 pM to about 350 pM, about 80 pM to about 300 pM, about 80 pM to about 250 pM, about 80 pM to about 200 pM, about 80 pM to about 150 pM, about 80 pM to about 100 pM, about 80 pM to about 90 pM, about 90 pM to about 30 nM, about 90 pM to about 25 nM, about 90 pM to about 30 nM, about 90 pM to about 15 nM, about 90 pM to about 10 nM, about 90 pM to about 5 nM, about 90 pM to about 2 nM, about 90 pM to about 1 nM, about 90 pM to about 950 pM, about 90 pM to about 900 pM, about 90 pM to about 850 pM, about 90 pM to about 800 pM, about 90 pM to about 750 pM, about 90 pM to about 700 pM, about 90 pM to about 650 pM, about 90 pM to about 600 pM, about 90 pM to about 550 pM, about 90 pM to about 500 pM, about 90 pM to about 450 pM, about 90 pM to about 400 pM, about 90 pM to about 350 pM, about 90 pM to about 300 pM, about 90 pM to about 250 pM, about 90 pM to about 200 pM, about 90 pM to about 150 pM, about 90 pM to about 100 pM, about 100 pM to about 30 nM, about 100 pM to about 25 nM, about 100 pM to about 30 nM, about 100 pM to about 15 nM, about 100 pM to about 10 nM, about 100 pM to about 5 nM, about 100 pM to about 2 nM, about 100 pM to about 1 nM, about 100 pM to about 950 pM, about 100 pM to about 900 pM, about 100 pM to about 850 pM, about 100 pM to about 800 pM, about 100 pM to about 750 pM, about 100 pM to about 700 pM, about 100 pM to about 650 pM, about 100 pM to about 600 pM, about 100 pM to about 550 pM, about

100 pM to about 500 pM, about 100 pM to about 450 pM, about 100 pM to about 400 pM, about 100 pM to about 350 pM, about 100 pM to about 300 pM, about 100 pM to about 250 pM, about 100 pM to about 200 pM, about 100 pM to about 150 pM, about 150 pM to about 30 nM, about 150 pM to about 25 nM, about 150 pM to about 30 nM, about 150 pM to about 15 nM, about 150 pM to about 10 nM, about 150 pM to about 5 nM, about 150 pM to about 2 nM, about 150 pM to about 1 nM, about 150 pM to about 950 pM, about 150 pM to about 900 pM, about 150 pM to about 850 pM, about 150 pM to about 800 pM, about 150 pM to about 750 pM, about 150 pM to about 700 pM, about 150 pM to about 650 pM, about 150 pM to about 600 pM, about 150 pM to about 550 pM, about 150 pM to about 500 pM, about 150 pM to about 450 pM, about 150 pM to about 400 pM, about 150 pM to about 350 pM, about 150 pM to about 300 pM, about 150 pM to about 250 pM, about 150 pM to about 200 pM, about 200 pM to about 30 nM, about 200 pM to about 25 nM, about 200 pM to about 30 nM, about 200 pM to about 15 nM, about 200 pM to about 10 nM, about 200 pM to about 5 nM, about 200 pM to about 2 nM, about 200 pM to about 1 nM, about 200 pM to about 950 pM, about 200 pM to about 900 pM, about 200 pM to about 850 pM, about 200 pM to about 800 pM, about 200 pM to about 750 pM, about 200 pM to about 700 pM, about 200 pM to about 650 pM, about 200 pM to about 600 pM, about 200 pM to about 550 pM, about 200 pM to about 500 pM, about 200 pM to about 450 pM, about 200 pM to about 400 pM, about 200 pM to about 350 pM, about 200 pM to about 300 pM, about 200 pM to about 250 pM, about 300 pM to about 30 nM, about 300 pM to about 25 nM, about 300 pM to about 30 nM, about 300 pM to about 15 nM, about 300 pM to about 10 nM, about 300 pM to about 5 nM, about 300 pM to about 2 nM, about 300 pM to about 1 nM, about 300 pM to about 950 pM, about 300 pM to about 900 pM, about 300 pM to about 850 pM, about 300 pM to about 800 pM, about 300 pM to about 750 pM, about 300 pM to about

700 pM, about 300 pM to about 650 pM, about 300 pM to about 600 pM, about 300 pM to about 550 pM, about 300 pM to about 500 pM, about 300 pM to about 450 pM, about 300 pM to about 400 pM, about 300 pM to about 350 pM, about 400 pM to about 30 nM, about 400 pM to about 25 nM, about 400 pM to about 30 nM, about 400 pM to about 15 nM, about 400 pM to about 10 nM, about 400 pM to about 5 nM, about 400 pM to about 2 nM, about 400 pM to about 1 nM, about 400 pM to about 950 pM, about 400 pM to about 900 pM, about 400 pM to about 850 pM, about 400 pM to about 800 pM, about 400 pM to about 750 pM, about 400 pM to about 700 pM, about 400 pM to about 650 pM, about 400 pM to about 600 pM, about 400 pM to about 550 pM, about 400 pM to about 500 pM, about 500 pM to about 30 nM, about 500 pM to about 25 nM, about 500 pM to about 30 nM, about 500 pM to about 15 nM, about 500 pM to about 10 nM, about

500 pM to about 5 nM, about 500 pM to about 2 nM, about 500 pM to about 1 nM, about 500 pM to about 950 pM, about 500 pM to about 900 pM, about 500 pM to about 850 pM, about 500 pM to about 800 pM, about 500 pM to about 750 pM, about 500 pM to about 700 pM, about 500 pM to about 650 pM, about 500 pM to about 600 pM, about 500 pM to about 550 pM, about 600 pM to about 30 nM, about 600 pM to about 25 nM, about 600 pM to about 30 nM, about 600 pM to about 15 nM, about 600 pM to about 10 nM, about 600 pM to about 5 nM, about 600 pM to about 2 nM, about 600 pM to about 1 nM, about 600 pM to about 950 pM, about 600 pM to about 900 pM, about 600 pM to about 850 pM, about 600 pM to about 800 pM, about 600 pM to about 750 pM, about 600 pM to about 700 pM, about 600 pM to about 650 pM, about 700 pM to about 30 nM, about 700 pM to about 25 nM, about 700 pM to about 30 nM, about 700 pM to about 15 nM, about 700 pM to about 10 nM, about 700 pM to about 5 nM, about 700 pM to about 2 nM, about 700 pM to about 1 nM, about 700 pM to about 950 pM, about 700 pM to about 900 pM, about 700 pM to about 850 pM, about 700 pM to about 800 pM, about 700 pM to about 750 pM, about 800 pM to about 30 nM, about 800 pM to about 25 nM, about 800 pM to about 30 nM, about 800 pM to about 15 nM, about 800 pM to about 10 nM, about 800 pM to about 5 nM, about 800 pM to about 2 nM, about 800 pM to about 1 nM, about 800 pM to about 950 pM, about 800 pM to about 900 pM, about 800 pM to about 850 pM, about 900 pM to about 30 nM, about 900 pM to about 25 nM, about 900 pM to about 30 nM, about 900 pM to about 15 nM, about 900 pM to about 10 nM, about

900 pM to about 5 nM, about 900 pM to about 2 nM, about 900 pM to about 1 nM, about 900 pM to about 950 pM, about 1 nM to about 30 nM, about 1 nM to about 25 nM, about 1 nM to about 20 nM, about 1 nM to about 15 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, about 2 nM to about 30 nM, about 2 nM to about 25 nM, about 2 nM to about 20 nM, about 2 nM to about 15 nM, about 2 nM to about 10 nM, about 2 nM to about 5 nM, about 4 nM to about 30 nM, about 4 nM to about 25 nM, about 4 nM to about 20 nM, about 4 nM to about 15 nM, about 4 nM to about 10 nM, about 4 nM to about 5 nM, about 5 nM to about 30 nM, about 5 nM to about 25 nM, about 5 nM to about 20 nM, about 5 nM to about 15 nM, about 5 nM to about 10 nM, about 10 nM to about 30 nM, about 10 nM to about 25 nM, about 10 nM to about 20 nM, about 10 nM to about 15 nM, about 15 nM to about 30 nM, about 15 nM to about 25 nM, about 15 nM to about 20 nM, about 20 nM to about 30 nM, and about 20 nM to about 25 nM).

Any of the target-binding domains described herein can bind to its target with a KD of between about 1 nM to about 10 nM (e.g., about 1 nM to about 9 nM, about 1 nM to about 8 nM, about 1 nM to about 7 nM, about 1 nM to about 6 nM, about 1 nM to about 5 nM, about 1 nM to about 4 nM, about 1 nM to about 3 nM, about 1 nM to about 2 nM, about 2 nM to about 10 nM, about 2 nM to about 9 nM, about 2 nM to about 8 nM, about 2 nM to about 7 nM, about 2 nM to about 6 nM, about 2 nM to about 5 nM, about 2 nM to about 4 nM, about 2 nM to about 3 nM, about 3 nM to about 10 nM, about 3 nM to about 9 nM, about 3 nM to about 8 nM, about 3 nM to about 7 nM, about 3 nM to about 6 nM, about 3 nM to about 5 nM, about 3 nM to about 4 nM, about 4 nM to about 10 nM, about 4 nM to about 9 nM, about 4 nM to about 8 nM, about 4 nM to about 7 nM, about 4 nM to about 6 nM, about 4 nM to about 5 nM, about 5 nM to about 10 nM, about 5 nM to about 9 nM, about 5 nM to about 8 nM, about 5 nM to about 7 nM, about 5 nM to about 6 nM, about 6 nM to about 10 nM, about 6 nM to about 9 nM, about 6 nM to about 8 nM, about 6 nM to about 7 nM, about 7 nM to about 10 nM, about 7 nM to about 9 nM, about 7 nM to about 8 nM, about 8 nM to about 10 nM, about 8 nM to about 9 nM, and about 9 nM to about 10 nM).

A variety of different methods known in the art can be used to determine the KD values of any of the antigen-binding protein constructs described herein (e.g., an electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.). Antigen-Binding Domains

In some embodiments of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second target-binding domain bind specifically to the same antigen. In some embodiments of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second target-binding domain bind specifically to different antigens.

In some embodiments of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein, one or both of the first target- binding domain and the second target-binding domain is an antigen-binding domain. In some embodiments of any of the single-chain chimeric polypeptides or any of the multi- chain chimeric polypeptides described herein, the first target-binding domain and the second target-binding domain are each an antigen-binding domain.

In some embodiments of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein, the antigen-binding domain includes or is a scFv or a single domain antibody (e.g., a VHH or a VNAR domain).

In some examples, an antigen-binding domain (e.g., any of the antigen-binding domains described herein) can bind specifically to any one of CDl6a (see, e.g., those described in U.S. Patent No. 9,035,026), CD28 (see, e.g., those described in U.S. Patent No.

7,723,482), CD3 (see, e.g., those described in U.S. Patent No. 9,226,962), CD33 (see, e.g., those described in U.S. Patent No. 8,759,494), CD20 (see, e.g., those described in WO 2014/026054), CD19 (see, e.g., those described in U.S. Patent No. 9,701,758), CD22 (see, e.g., those described in WO 2003/104425), CD123 (see, e.g., those described in WO 2014/130635), IL-1R (see, e.g., those described in U.S. Patent No. 8,741,604), IL-l (see, e.g., those described in WO 2014/095808), VEGF (see, e.g., those described in U.S. Patent No. 9,090,684), IL-6R (see, e.g., those described in U.S. Patent No. 7,482,436), IL-4 (see, e.g., those described in U.S. Patent Application Publication No.

2012/0171197), IL-10 (see, e.g., those described in U.S. Patent Application Publication No. 2016/0340413), PDL-l (see, e.g., those described in Drees et al., Protein Express. Purif 94:60-66, 2014), TIGIT (see, e.g., those described in U.S. Patent Application Publication No. 2017/0198042), PD-l (see, e.g., those described in U.S. Patent No.

7,488,802), TIM3 (see, e.g., those described in U.S. Patent No. 8,552,156), CTLA4 (see, e.g., those described in WO 2012/120125), MICA (see, e.g., those described in WO 2016/154585), MICB (see, e.g., those described in U.S. Patent No. 8,753,640), IL-6 (see, e.g., those described in Gejima et al., Human Antibodies 11 (4) : 121 - 129, 2002), IL-8 (see, e.g., those described in U.S. Patent No. 6,117,980), TNFa (see, e.g., those described in Geng et al., Immunol. Res. 62(3):377-385, 2015), CD26a (see, e.g., those described in WO 2017/189526), CD36 (see, e.g., those described in U.S. Patent Application

Publication No. 2015/0259429), ULBP2 (see, e.g., those described in U.S. Patent No. 9,273,136), CD30 (see, e.g., those described in Homach et al., Scand. J. Immunol.

48(5):497-50l, 1998), CD200 (see, e.g., those described in U.S. Patent No. 9,085,623), IGF-1R (see, e.g., those described in U.S. Patent Application Publication No.

2017/0051063), MUC4AC (see, e.g., those described in WO 2012/170470), MUC5AC (see, e.g., those described in U.S. Patent No. 9,238,084), Trop-2 (see, e.g., those described in WO 2013/068946), CMET (see, e.g., those described in Edwardraja et al., Biotechnol. Bioeng. l06(3):367-375, 2010), EGFR (see, e.g., those described in Akbari et al., Protein Expr. Purif. 127:8-15, 2016), HER1 (see, e.g., those described in U.S. Patent Application Publication No. 2013/0274446), HER2 (see, e.g., those described in Cao et al., Biotechnol. Lett. 37(7):l347-l354, 2015), HER3 (see, e.g., those described in U.S. Patent No. 9,505,843), PSMA (see, e.g., those described in Parker et al., Protein Expr. Purif. 89(2): 136-145, 2013), CEA (see, e.g., those described in WO 1995/015341), B7H3 (see, e.g., those described in U.S. Patent No. 9,371,395), EPCAM (see, e.g., those described in WO 2014/159531), BCMA (see, e.g., those described in Smith et al., Mol. Ther. 26(6): 1447-1456, 2018), P-cadherin (see, e.g., those described in U.S. Patent No. 7,452,537), CEACAM5 (see, e.g., those described in U.S. Patent No. 9,617,345), a

ULl6-binding protein (see, e.g., those described in WO 2017/083612), HLA-DR (see, e.g., Pistillo et al., Exp. Clin. Immunogenet. 14(2): 123-130, 1997), DLL4 (see, e.g., those described in WO 2014/007513), TYR03 (see, e.g., those described in WO 2016/166348), AXL (see, e.g., those described in WO 2012/175692), MER (see, e.g., those described in WO 2016/106221), CD122 (see, e.g., those described in U.S. Patent Application

Publication No. 2016/0367664), CD155 (see, e.g., those described in WO 2017/149538), and PDGF-DD (see, e.g., those described in U.S. Patent No. 9,441,034). The antigen binding domains present in any of the single-chain chimeric polypeptides or any of the multi-chain polypeptides described herein are each independently selected from the group consisting of: a VHH domain, a VNAR domain, and a scFv.

In some embodiments, the first target binding domain, the second target-binding domain, and/or one or more of the one or more additional antigen-binding domains can be an anti-CD3 scFv. In some embodiments, the anti-CD3 scFv can include a heavy chain variable domain including a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to

QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINP SRGYTNYN QKFKDK ATLTTDK S S S T A YMQL S SLT SED S A V Y Y C ARY YDDH Y CL DYWGQGTTLTVSS (SEQ ID NO: 16) and/or a light chain variable domain including a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to

QIVLT Q SP AIMS ASPGEK VTMT C S AS S S VS YMNW Y QQK SGTSPKRWI YDT SKL A SGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR

(SEQ ID NO: 17). In some embodiments, a scFv (e.g., any of the scFvs described herein) can include a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the heavy chain variable domain and the light chain variable domain. In some embodiments, the anti-CD3 scFv can include a heavy chain variable domain encoded by a nucleic acid including a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to CAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCTTCCG TGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAATGCAC TGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATCAACC CCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCACCCT CACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTTAACA TCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATTACTG CCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC (SEQ ID NO: 18), and/or a light chain variable domain encoded by a nucleic acid including a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to

CAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAA AGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTA TCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCAGCAA GCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAACAAGC TACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATTACT GTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTCGA GATTAATCGT (SEQ ID NO: 19).

In some embodiments, an anti-CD3 scFv can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to QIVLT Q SP AIMS ASPGEK VTMT C S AS S S VS YMNW Y QQK SGTSPKRWIYDT SKL A S GVP AHFRGS GS GT S Y SLTI S GME AED A AT Y Y C QQ W S SNPFTF GS GTKLEINRGG GGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQ RPGQGLEWIGYINP SRGYTNYN QKFKDK ATLTTDK S S S T A YMQL S SLT SED S A V YY C ARYYDDH Y CLD YW GQGTTLT V S S (SEQ ID NO: 20). In some embodiments, an anti-CD3 scFv can include the six CDRs present in SEQ ID NO: 20.

In some embodiments, an anti-CD3 scFv can include a sequence encoded by a nucleic acid sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to CAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAA AGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTA TCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCAGCAA GCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAACAAGC TACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATTACT GTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTCGA GAT T A AT C GT GG AGGC GG AGGT AGC GG AGG AGGC GG AT C C GGC GGT GG AGG TAGCCAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCT TCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAAT GCACTGGGTCAAGC AACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATC AACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCA CCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTT AACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATT ACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC (SEQ ID NO: 21).

In some embodiments, the first target binding domain, the second target-binding domain, and/or one or more of the one or more additional antigen-binding domains can be an anti-CD28 scFv. In some embodiments, the anti-CD28 scFv can include a heavy chain variable domain including a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to

DIEMTQ SP AIMS ASLGERVTMTCT AS S S VS S S YFHW YQQKPGS SPKLCIYST SNL A S GVPPRF SGSGSTSYSLTIS SME AED A AT YF CHQ YHRSPTF GGGTKLETKR (SEQ ID NO: 22) and/or a light chain variable domain including a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYN D YTKYNEKFKGK ATLT SDKS SIT AYMEF S SLT SED S AL Y Y C ARW GDGNYW GRG TTLTVSS (SEQ ID NO: 23). In some embodiments, the anti-CD28 scFv can include a heavy chain variable domain encoded by a nucleic acid including a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to

GACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCTCTTTAGGAGAAC GTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTTCCAC TGGTACCAGCAGAAACCCGGCTCCTCCCCTAAACTGTGTATCTACTCCACAA GCAATTTAGCTAGCGGCGTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACCTCT TACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTTTTG CCATCAGTACCACCGGTCCCCTACCTTTGGCGGAGGCACAAAGCTGGAGACC AAGCGG (SEQ ID NO: 24), and/or a light chain variable domain encoded by a nucleic acid including a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to

GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGA AAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGG GT GAAGC AGA AGCCCGGT C AAGGTCTCGAGT GGATCGGC AGC AT C AATCCCT ACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGAC AAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCTG AGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGGC CGGGGAACTACTTTAACAGTGAGCTCC (SEQ ID NO: 25).

In some embodiments, an anti-CD28 scFv can include a sequence that is at least

70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYN D YTKYNEKFKGK ATLT SDKS SIT AYMEF S SLT SED S AL Y Y C ARW GDGNYW GRG TTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSY FHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCH Q YHRSPTF GGGTKLETKR (SEQ ID NO: 26). In some embodiments, an anti-CD28 scFv can include the six CDRs present in SEQ ID NO: 26.

In some embodiments, an anti-CD28 scFv can include a sequence encoded by a nucleic acid sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to

GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGA

AAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGG

GT GAAGC AGA AGCCCGGT C AAGGTCTCGAGT GGATCGGC AGC AT C AATCCCT

ACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGAC

AAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCTG

AGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGGC

CGGGGAACTACTTTAACAGTGAGCTCCGGCGGCGGCGGAAGCGGAGGTGGA

GGATCTGGCGGTGGAGGCAGCGACATCGAGATGACACAGTCCCCCGCTATCA

TGAGCGCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCCAGC

GTGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTAA

ACTGTGTATCTACTCCACAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTTA

GCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCGAA

GATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGG

AGGCACAAAGCTGGAGACCAAGCGG (SEQ ID NO: 27).

In some embodiments, any of the antigen-binding domains described herein is a BiTe, a (scFv)2, a nanobody, a nanobody -HS A, a DART, a TandAb, a scDiabody, a scDiabody-CEB, scFv-CH-CL-scFv, a HSAbody, scDiabody-HAS, or a tandem-scFv. Additional examples of antigen-binding domains that can be used in any of the single chain chimeric polypeptides or multi-chain chimeric polypeptides described herein are known in the art.

A VHH domain is a single monomeric variable antibody domain that can be found in camelids. A VNAR domain is a single monomeric variable antibody domain that can be found in cartilaginous fish. Non -limiting aspects of VHH domains and VNAR domains are described in, e.g., Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016; De Genst et al., Dev. Comp. Immunol. 30: 187-198, 2006; De Meyer et al., Trends

Biotechnol. 32:263-270, 2014; Kijanka et al., Nanomedicine 10: 161-174, 2015; Kovaleva et al., Expert. Opin. Biol. Ther. 14: 1527-1539, 2014; Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delic et ak, Trends Pharmacol. Sci. 35:247-255, 2014; Muyldermans, J. Biotechnol. 74:277-302, 2001; Muyldermans et al., Trends Biochem. Sci. 26:230-235, 2001; Muyldermans, Ann. Rev. Biochem. 82:775-797, 2013; Rahbarizadeh et al., Immunol. Invest. 40:299-338, 2011; Van Audenhove et al.,

EBioMedicine 8:40-48, 2016; Van Bockstaele et al., Curr. Opin. Investig. Drugs 10: 1212- 1224, 2009; Vincke et al., Methods Mol. Biol. 911 : 15-26, 2012; and Wesolowski et al., Med. Microbiol. Immunol. 198: 157-174, 2009.

In some embodiments, each of the antigen-binding domains in the single-chain chimeric polypeptides or multi-chain chimeric polypeptides described herein are both VHH domains, or at least one antigen-binding domain is a VHH domain. In some embodiments, each of the antigen-binding domains in the single-chain chimeric polypeptides or multi-chain chimeric polypeptides described herein are both VNAR domains, or at least one antigen-binding domain is a VNAR domain. In some

embodiments, each of the antigen-binding domains in the single-chain chimeric polypeptides or multi-chain chimeric polypeptides described herein are both scFv domains, or at least one antigen-binding domain is a scFv domain. DARTs are described in, e.g., Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

In some embodiments of any of the antigen-binding domains described herein, the antigen-binding domain can bind to an antigen selected from the group consisting of: a protein, a carbohydrate, a lipid, and a combination thereof.

Additional examples and aspects of antigen-binding domains are known in the art.

Soluble Interleukin or Cytokine Protein

In some embodiments of any of the single-chain chimeric polypeptides or multi chain chimeric polypeptides described herein, one or both of the first target-binding domain and the second target-binding domain can be a soluble interleukin protein, soluble cytokine protein, or soluble cell surface protein. In some embodiments, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group of: IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein (e g., ULBP1, ULBP2, ULBP3, ULBP4, ULPB5, or ULBP6. Non-limiting examples of soluble IL-2, IL-3, IL-7, IL-8, IL-10, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF and FLT3L.. Human Soluble IL-2 (SEQ ID NO: 28)

APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTEMCEYADE TATIVEFLNR WITFCQSIIS TLT

Human Soluble IL-3 (SEQ ID NO: 29)

APMTQTTPLKT SWVNCSNMID El ITHLKQPP LPLLDFNNLN GEDQDILMEN NLRRPNLEAF NRAVKSLQNA SAIESILKNL LPCLPLATAA PTRHPIHIKD GDWNEFRRKL TFYLKTLENA QAQQTTLSLA IF

Human Soluble IL-8 (SEQ ID NO: 31)

EGAVLPRSAK ELRCQCIKTY SKPFHPKFIK ELRVIESGPH CANTEI IVKL SDGRELCLDP KENWVQRWE KFLKRAENS

Human Soluble IL-10 (SEQ ID NO: 32)

SPGQGTQSENSC THFPGNLPNM LRDLRDAFSR VKTFFQMKDQ LDNLLLKESL LEDFKGYLGC QALSEMIQFY LEEVMPQAEN QDPDIKAHVN SLGENLKTLR LRLRRCHRFL PCENKSKAVE QVKNAFNKLQ EKGIYKAMSE FDIFINYIEA YMTMKIRN

Human Soluble IL-12P (p40) (SEQ ID NO: 33)

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKEF GD AGQ YTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQGVTCGAATLS AERVRGDNK EYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNL QLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTS AT VICRKNASIS VRAQDRYYS S SWSEWAS VPCS

Nucleic Acid Encoding Human Soluble IL-12P (p40) (SEQ ID NO: 34)

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG

ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC

CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAA

GGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACG

GAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGAC

CTTTTTAAGGTGT GAGGCC AAAAACTAC AGCGGTCGTTT C ACTT GTT GGT GGC

TGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGC

TCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGG

TTCGT GGCGATAAC AAGGAATACGAGTAC AGCGT GGAGT GCC AAGAAGATAG

CGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCG

TGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATC

ATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCG

GCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCT

ACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAG

AAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGA

ACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCC

GAGTGGGCCAGCGTGCCTTGTTCC

Human Soluble IL-12a (p35) (SEQ ID NO: 35)

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKD KT ST VE ACLPLELTKNE S CLN SRET SFITN GSCL ASRKT SFMM ALCL S SI YEDLKM Y Q VEFKTMNAKLLMDPKRQIFLDQNML AVIDELMQ ALNFN SET VPQK S SLEEPD FYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

Nucleic Acid Encoding Human Soluble IL-12a (p35) (SEQ ID NO: 36)

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCAC

AGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGA

CTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCA

AGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAA

CGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTT

AGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGA

GGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCA T GGACCCTA AACGGC AGATCTTTTTAGACC AGAAC AT GCTGGCTGT GATTGAT

GAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTC

CCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCC

ACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAAC

GCCAGC

Exemplary Human Soluble IL-12 (SEQ ID NO: 37)

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLT

IQVKEF GD AGQ YTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKNKTFLR

CEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQGVTCGAATLS AERVRGDN

KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKN

LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT

SATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPV

ATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTV

EACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEF

KTMNAKLLMDPKRQIFLDQNMLAVIDELMQ ALNFN SETVPQKS SLEEPDFYKTK

IKLCILLHAFRIRAVTIDRVMSYLNAS

Nucleic Acid Encoding Exemplary Human Soluble IL-12 (SEQ ID NO: 38)

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG

ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG

CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC

CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA

AGGGAGGCGAGGT GCTC AGCC ATTCCTT ATT ATT ATT AC AC AAGAAGGAAGA

CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG

ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG

GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA

AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA

GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG

ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC

GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA

GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA

CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG

AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG

GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGC

TGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAG

GTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCC

CGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGC

AACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCG

AGGAGATCGACC AT GAAGAT AT C ACC AAGGAC AAGAC ATCC ACCGT GGAGG

CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAA

ACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTAT

GATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGG

AGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTT

TTT AGACC AGAAC AT GCTGGCTGT GATT GAT GAGCTGATGC AAGCTTT AAACT

TCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTAC

AAGAC AAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCG

TGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC

Human Soluble IL-15 (SEQ ID NO: 39)

NWVNVISDLKKI EDLIQSMHID ATLYTESDVH PSCKVTAMKC FLLELQVISL ESGDASIHDT VENLI ILANN SLSSNGNVTE SGCKECEELE EKNIKEFLQS FVHIVQMFIN TS

Human Soluble IL-17 (SEQ ID NO: 40)

GITIPRN PGCPNSEDKN FPRTVMVNLN IHNRNTNTNP KRSSDYYNRS TSPWNLHRNE DPERYPSVIW EAKCRHLGCI NADGNVDYHM NSVPIQQEIL

VLRREPPHCP NSFRLEKILV SVGCTCVTPI VHHVA Human Soluble IL-18 (SEQ ID NO: 41)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFI ISMYKDSQPRG MAVTISVKCEKISTLSCENKI ISFKEMNPPDNIKDTKSDI IFFQRSVPGHDNKMQFESS SYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

Nucleic Acid Encoding Human Soluble IL-18 (SEQ ID NO: 42)

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCA

AGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACT

CCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGG

ACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAAT

CAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCC

CCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGC

CCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTT

TAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGA

CGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT

Human Soluble PDGF-DD (SEQ ID NO: 45)

RDTSATPQSASI KALRNANLRR DESNHLTDLY RRDETIQVKG NGYVQSPRFP NSYPRNLLLT WRLHSQENTR IQLVFDNQFG LEEAENDICR YDFVEVEDIS ETSTI IRGRW CGHKEVPPRI KSRTNQIKIT FKSDDYFVAK PGFKIYYSLL EDFQPAAASE TNWESVTSSI SGVSYNSPSV TDPTLIADAL DKKIAEFDTV EDLLKYFNPE SWQEDLENMY LDTPRYRGRS YHDRKSKVDL DRLNDDAKRY SCTPRNYSVN IREELKLANV VFFPRCLLVQ RCGGNCGCGT VNWRSCTCNS GKTVKKYHEV LQFEPGHIKR RGRAKTMALV DIQLDHHERC DCICSSRPPR

Human Soluble SCF (SEQ ID NO: 46)

EGICRNRVTNNVKDV TKLVANLPKD YMITLKYVPG MDVLPSHCWI SEMWQLSDS LTDLLDKFSN ISEGLSNYSI IDKLVNIVDD LVECVKENSS KDLKKSFKSP EPRLFTPEEF FRIFNRSIDA FKDFWASET SDCWSSTLS PEKDSRVSVT KPEMLPPVAA SSLRNDSSSS NRKAKNPPGD SSLHWAAMAL PALFSLI IGF AFGALYWKKR QPSLTRAVEN IQINEEDNEI SMLQEKEREF QEV Human Soluble FLT3L (SEQ ID NO: 47)

tqdcsfqhspissd favkirelsd yllqdypvtv asnlqdeelc gglwrlvlaq rwmerlktva gskmqgller vnteihfvtk cafqpppscl rfvqtnisrl lqetseqlva lkpwitrqnf srclelqcqp dsstlpppws prpleatapt apqpplllll llpvglllla aawclhwqrt rrrtprpgeq vppvpspqdl llveh

Exemplary soluble cell surface proteins include soluble MICA, MICB, and a ULP16 binding protein (e g., ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, or ULBP6) Exemplary sequences for soluble MICA, MICB, EILBP1, EILBP2, EILBP3, EiLBP4, EILBP5, and EILBP6 are listed below.

Human Soluble MICA (SEQ ID NO: 48)

ephslry nltvlswdgs vqsgfltevh ldgqpflrcd rqkcrakpqg qwaedvlgnk twdretrdlt gngkdlrmtl ahikdqkegl hslqeirvce ihednstrss qhfyydgelf lsqnletkew tmpqssraqt lamnvrnflk edamktkthy hamhadclqe lrrylksgvv lrrtvppmvn vtrseasegn itvtcrasgf ypwnitlswr qdgvslshdt qqwgdvlpdg ngtyqtwvat ricqgeeqrf tcymehsgnh sthpvpsgkv lvlqshwqtf hvsavaaaai fviiifyvrc ckkktsaaeg pelvslqvld qhpvgtsdhr datqlgfqpl msdlgstgst ega

Human Soluble MICB (SEQ ID NO: 49)

aephslry nlmvlsqdes vqsgflaegh ldgqpflryd rqkrrakpqg qwaedvlgak twdtetedlt engqdlrrtl thikdqkggl hslqeirvce ihedsstrgs rhfyydgelf lsqnletqes tvpqssraqt lamnvtnfwk edamktkthy ramqadclqk lqrylksgva irrtvppmvn vtcsevsegn itvtcrassf yprnitltwr qdgvslshnt qqwgdvlpdg ngtyqtwvat rirqgeeqrf tcymehsgnh gthpvpsgkv lvlqsqrtdf pyvsaampcf viiiilcvpc ckkktsaaeg pelvslqvld qhpvgtgdhr

daaqlgfqpl msatgstgst ega Human Soluble ULBP1 (SEQ ID NO: 50)

wvdthclcydfiit pksrpepqwc evqglvderp flhydcvnhk akafaslgkk vnvtktweeq tetlrdvvdf lkgqlldiqv enlipieplt lqarmscehe ahghgrgswq flfngqkfll fdsnnrkwta lhpgakkmte kweknrdvtm ffqkislgdc kmwleeflmy weqmldptkp pslapg

Human Soluble ULBP2 (SEQ ID NO: 51)

gradphslcyditvi pkfrpgprwc avqgqvdekt flhydcgnkt vtpvsplgkk lnvttawkaq npvlrevvdi lteqlrdiql enytpkeplt lqarmsceqk aeghssgswq fsfdgqifll fdsekrmwtt vhpgarkmke kwendkvvam sfhyfsmgdc igwledflmg mdstlepsag aplams

Human Soluble ULBP3 (SEQ ID NO: 52)

dahslwynfti ihlprhgqqw cevqsqvdqk nflsydcgsd kvlsmghlee qlyatdawgk qlemlrevgq rlrleladte ledftpsgpl tlqvrmscec eadgyirgsw qfsfdgrkfl lfdsnnrkwt vvhagarrmk ekwekdsglt tffkmvsmrd ckswlrdflm hrkkrlepta pptmapg

Human Soluble ULBP4 (SEQ ID NO: 53)

hslcfnftik slsrpgqpwc eaqvflnknl flqynsdnnm vkplgllgkk vyatstwgel tqtlgevgrd lrmllcdikp qiktsdpstl qvemfcqrea erctgaswqf atngeksllf damnmtwtvi nheaskiket wkkdrgleky frklskgdcd hwlreflghw eampeptvsp vnasdihwss sslpdrwiil gafillvlmg ivlicvwwqn gewqaglwpl rts

Human Soluble ULBP5 (SEQ ID NO: 54)

gladp hslcyditvi pkfrpgprwc avqgqvdekt flhydcgskt

vtpvsplgkk lnvttawkaq npvlrevvdi lteqlldiql enyipkeplt lqarmsceqk aeghgsgswq lsfdgqifll fdsenrmwtt vhpgarkmke kwendkdmtm sfhyismgdc tgwledflmg mdstlepsag apptmssg Human Soluble ULBP6 (SEQ ID NO: 55)

rrddp hslcyditvi pkfrpgprwc avqgqvdekt flhydcgnkt

vtpvsplgkk lnvtmawkaq npvlrevvdi lteqlldiql enytpkeplt lqarmsceqk aeghssgswq fsidgqtfll fdsekrmwtt vhpgarkmke kwendkdvam sfhyismgdc igwledflmg mdstlepsag aplamssg

In some embodiments, a soluble IL-12 protein can include a first sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 33, and a second sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 35. In some embodiments, the soluble IL-12 can further include a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first sequence and the second sequence.

In some embodiments, a soluble IL-12 protein is encoded by a first nucleic acid encoding a first sequence at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 34, and a second nucleic acid sequence encoding a second sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 36. In some embodiments, the nucleic acid encoding a soluble IL-12 protein further includes a nucleic acid sequence encoding a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first nucleic acid and the second nucleic acid.

In some embodiments, a soluble IL-12 protein includes a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 37. In some embodiments, a soluble IL-12 protein is encoded by a nucleic acid including a sequence at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 38.

In some embodiments, a soluble IL-18 protein can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 41. In some embodiments, a soluble IL-18 protein is encoded by a nucleic acid including a sequence at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 42.

Additional examples of soluble interleukin proteins and soluble cytokine proteins are known in the art.

Soluble Interleukin or Cytokine Receptor

In some embodiments of any of the single-chain chimeric polypeptides or multi - chain chimeric polypeptides described herein, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin receptor or a soluble cytokine receptor. In some embodiments, the soluble receptor is a soluble TGF-b receptor II (TGF-b RII) (see, e.g., those described in Yung et al., Am. J. Resp. Crit. Care Med. 194(9): 1140-1151, 2016), a soluble TGF-b RIII (see, e.g., those described in Heng et al., Placenta 57:320, 2017), a soluble NKG2D (see, e.g., Cosman et al., Immunity

14(2): 123-133, 2001; Costa et al, Front. Immunol ., Vol. 9, Article 1150, May 29, 2018; doi: l0.3389/fimmu.20l8.0H50), a soluble NKp30 (see, e.g., Costa et al., Front.

Immunol ., Vol. 9, Article 1150, May 29, 2018; doi: l0.3389/fimmu.20l8.0l 150), a soluble NKp44 (see, e.g., those described in Costa et al., Front. Immunol ., Vol. 9, Article 1150, May 29, 2018; doi: l0.3389/fimmu.20l8.0l 150), a soluble NKp46 (see, e.g.,

Mandelboim et al., Nature 409:1055-1060, 2001; Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi: l0.3389/fimmu.20l8.0l 150), a soluble DNAM-l (see, e.g., those described in Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi: l0.3389/fimmu.20l8.0H50), a scMHCI (see, e.g., those described in Washburn et al., PLoS One 6(3):el8439, 2011), a scMHCII (see, e.g., those described in Bishwajit et al., Cellular Immunol. l70(l):25-33, 1996), a scTCR (see, e.g., those described in Weber et al., Nature 356(6372):793-796, 1992), a soluble CD155 (see, e.g., those described in Tahara-Hanaoka et al., Int. Immunol. l6(4):533-538, 2004), or a soluble CD28 (see, e.g., Hebbar et al., Clin. Exp. Immunol. 136:388-392, 2004).

In some embodiments, a soluble TGF RII receptor can include a first sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56), and a second sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVC VAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56). In some embodiments, the soluble TGF-b RII receptor can further include a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first sequence and the second sequence.

In some embodiments, a soluble TGF-b RII receptor is encoded by a first nucleic acid encoding a first sequence at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 57), and a second nucleic acid sequence encoding a second sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGC AGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 57). In some embodiments, the nucleic acid encoding a soluble TGF-b RII receptor further includes a nucleic acid sequence encoding a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first nucleic acid and the second nucleic acid.

In some embodiments, a soluble TGF-b RII receptor includes a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIF SEEYNT SNPD (SEQ ID NO: 60). In some embodiments, a soluble TGF-b RII receptor is encoded by a nucleic acid including a sequence at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATC ACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 61).

Additional examples of soluble interleukin receptors and soluble cytokine receptors are known in the art.

Ligands of Co-stimulatory Molecules

In some embodiments of any of the single-chain chimeric polypeptides or multi- chain chimeric polypeptides described herein, one or both of the first target-binding domain and the second target-binding domain is a ligand of a co-stimulatory molecule.

In some embodiments, the ligand of a co-stimulatory molecule is a soluble CD80 (see, e.g., those described in Haile et al., J Immunol. 2013 Sep 1;191(5):2829-36, and those described W02004/076488 Al), a soluble CD86 (see, e.g., those described in Jeannin et al., Immunity. 2000 Sep;l3(3):303-l2, and those described in WO 2004/076488 Al), a soluble CD40 (see, e.g., those described in Elgueta et al., Immunological Reviews ,

229(1), doi.org/l0. l l 1 l/j.l600-065X.2009.00782.x, and those described in WO

2001/083755 A2), a soluble ICOSL (see, e.g., those described in Chattopadyhay et al., J. Immunol ., September 15, 2006, 111 (6) 3920-3929), a soluble CD70 (see, e.g., those described in Miller et al., J. Neurosurg, 2010 Aug;l l3(2):280-5, and those described in US 2009/0148942 Al), a soluble OX40L (see, e.g., those described in Kondo et al., Hum. Immunol ., 2007 Jul;68(7):563-7l. Epub 2007 Apr 13), a soluble 4-1BBL (see, e.g., those described in Wang et al., Cancer Immunol Immunother., 2012 Apr;6l(4):489-95), a soluble GITRL (see, e.g., those described in Stone et al., J Virol., 2006 Feb;80(4): l762- 72), a soluble LIGHT (see, e.g., those described in Maeda et al., J. Immunol .. 2018 Jul 1;201(1):202-214), a soluble TIM3 (see, e.g., those described in Clayton et al., J. Virol.,

2015 Apr;89(7):3723-36), a soluble TIM4 (see, e.g., those described in Rhein et al., J. Virol., 2016 Jun 10;90(13):6097-6111), soluble a ICAM1 (see, e.g., those described in Witkowsa and Borawska, Eur Cytokine Netw. 2004 Apr-Jun;l5(2):9l-8), a soluble LFA3 (see, e.g., those described in Menshawy et al., Comparative Clinical Pathology, 27(3), 721-727, doi.org/l0. l007/s00580-0l8-2657-x), a soluble CDld (see, e.g., those described in Brennan et al., PNAS, August 1, 2017 114 (31) 8348-8353), or a soluble LLT-l (see, e.g., those described in Chalan et al., PLoS ONE, 10(7), e0l32436, doi.org/l0.l37l/journal.pone.0l32436).

In some embodiments, a soluble CD80, a soluble CD86, a soluble CD40, a soluble ICOSL, a soluble CD70, a soluble OX40L, a soluble 4-1BBL, a soluble GITRL, a soluble LIGHT, a soluble TIM3, a soluble TIM4, a soluble ICAM1, a soluble LFA3, a soluble CDld, or a soluble LLT-l can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the wild type sequence.

Additional examples of ligands of a co-stimulatory molecules are known in the art.

Additional Antigen-Binding Domains

Some embodiments of any of the single-chain chimeric polypeptides or multi- chain chimeric polypeptides described herein can further include one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) at its N- and/or C-terminus.

In some embodiments, the single-chain chimeric polypeptide or multi-chain chimeric polypeptide can include one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains (e.g., any of the exemplary target- binding domains described herein or known in the art) at its N-terminus. In some embodiments, one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) at the N- terminus of the single-chain chimeric polypeptide or multi-chain chimeric polypeptide can directly abut the first target-binding domain (e.g., any of the exemplary target- binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), or the linker domain (e.g., any of the exemplary linker domains described herein). In some embodiments, the single-chain chimeric polypeptide or multi-chain chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the at least one additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) at the N-terminus of the single-chain chimeric polypeptide and the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), or the linker domain (e.g., any of the exemplary linker domains described herein).

In some embodiments of any of the single-chain chimeric polypeptides or multi- chain chimeric polypeptide described herein, the polypeptide includes one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) at its C-terminus. In some embodiments, one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) at the C-terminus of the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide directly abuts the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), or the linker domain (e.g., any of the exemplary linker domains described herein or known in the art). In some embodiments, the single-chain chimeric polypeptide or multi-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the at least one additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) at the C-terminus of the single-chain chimeric polypeptide and the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), or the linker domain (e.g., any of the exemplary linker domains described herein).

In some embodiments of any of the single-chain chimeric polypeptides or multi - chain chimeric polypeptides described herein, the polypeptide comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) at its N-terminus and its C-terminus. In some embodiments, one of the one or more additional antigen binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) at the N-terminus of the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide directly abuts the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), or the linker domain (e.g., any of the exemplary linker domains described herein). In some embodiments, the single-chain chimeric polypeptide or multi-chain chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the one or more additional antigen-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) at the N- terminus and the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), or the linker domain (e.g., any of the exemplary linker domains). In some embodiments, one of the one or more additional antigen binding domains (e.g., any of the exemplary target- binding domains described herein or known in the art) at the C-terminus directly abuts the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), or the linker domain (e.g., any of the exemplary linker domains). In some embodiments, the single- chain chimeric polypeptide or multi-chain chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the one or more additional antigen-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) at the C-terminus and the first target-binding domain(e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), or the linker domain (e.g., any of the exemplary linker domains described herein).

In some embodiments of any of the single-chain chimeric polypeptides or multi- chain chimeric polypeptides described herein, two or more (e.g., three, four, five, six, seven, eight, nine, or ten) of the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), and the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) bind specifically to the same antigen. In some embodiments, two or more (e.g., three, four, five, six, seven, eight, nine, or ten) of the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), and the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) bind specifically to the same epitope. In some embodiments, two or more (e.g., three, four, five, six, seven, eight, nine, or ten) of the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), and the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) include the same amino acid sequence. In some embodiments of any of the single-chain chimeric polypeptides or multi- chain chimeric polypeptides described herein, the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), and the one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains (e.g., any of the exemplary target- binding domains described herein or known in the art) each bind specifically to the same antigen. In some embodiments, the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain(e.g., any of the exemplary target-binding domains described herein or known in the art), and the one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains (e.g., any of the exemplary target- binding domains described herein or known in the art) each bind specifically to the same epitope. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains each comprise the same amino acid sequence.

In some embodiments of any of the single-chain chimeric polypeptides or multi- chain chimeric polypeptides described herein, the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), and the one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains (e.g., any of the exemplary target- binding domains described herein or known in the art) bind specifically to different antigens.

In some embodiments of any of the single-chain chimeric polypeptides or multi- chain chimeric polypeptides described herein, one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen-binding domain (e.g., any of the exemplary antigen-binding domains described herein or known in the art). In some embodiments of any of the single-chain chimeric polypeptides or multi-chain chimeric polypeptides described herein, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen-binding domain (e.g., any of the exemplary antigen-binding domains described herein or known in the art). In some embodiments, the antigen-binding domain can include a scFv or a single domain antibody.

In some embodiments of any of the single-chain chimeric polypeptides or multi- chain chimeric polypeptides described herein, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) of the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), and the one or more target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20,

CD 19, CD22, CD40, CD47, CD52, CD70, CD80, CD86, CD123, CD137, CD272, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, PDL-2, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, B7-H4, HVEM, EPCAM, BCMA, P-cadherin, CEACAM5, VISTA, a UL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, ILT3, ILT4, TIGIT, MHCII, LAG3, 0X40, a ligand of TGF-b receptor II (TGF-pRII), a ligand of TGF-pRIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL- 17, a receptor for IL-l 8, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a EILP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.

In some embodiments of any of the single-chain chimeric polypeptides or multi chain chimeric polypeptides described herein, one or more of the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), and the one or more additional target- binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. Non-limiting examples of soluble interleukin proteins, soluble cytokine proteins, or soluble cell surface proteins include: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL- 17, IL- 18, IL-21 , PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP 16-binding protein.

In some embodiments of any of the single-chain chimeric polypeptides or multi chain chimeric polypeptides described herein, one or more of the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), and the one or more additional target binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) is a soluble interleukin or cytokine receptor. Non-limiting examples of soluble interleukin receptors and soluble cytokine receptors include: a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further includes one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domain(s) (e.g., any of the exemplary target-binding domains described herein or known in the art), where at least one of the one or more additional antigen-binding domain(s) is positioned between the linker domain (e.g., any of the exemplary linker domains described herein or known in the art) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein). In some embodiments, the first chimeric polypeptide of a multi-chain chimeric polypeptide can further include a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the linker domain (e.g., any of the exemplary linker domains described herein) and the at least one of the one or more additional target-binding domain(s) (e.g., any of the exemplary target-binding domains described herein or known in the art), and/or a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the at least one of the one or more additional target-binding domain(s) (e.g., any of the exemplary target- binding domains described herein or known in the art) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further includes one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) directly abuts the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide of a multi-chain chimeric polypeptide. In some embodiments, the first chimeric polypeptide of a multi-chain chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, the at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) directly abuts the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) in the first chimeric polypeptide of a multi- chain chimeric polypeptide. In some embodiments, the first chimeric polypeptide of a multi-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the at least one of the one or more additional target-binding domains (e.g., any of the exemplary target- binding domains described herein or known in the art) and the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art). In some embodiments of any of the multi-chain chimeric polypeptides described herein, at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) is positioned between the linker domain (e.g., any of the exemplary linker domains described herein or known in the art) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the at least one additional target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) or the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide of a multi-chain chimeric polypeptide. In some embodiments, the first chimeric polypeptide of a multi- chain chimeric polypeptide further comprises a linker sequence (e.g., any of the linker sequences described herein or known in the art) disposed between the at least one additional target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) and the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) or the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the at least one additional target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) or the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide of a multi-chain chimeric polypeptide. In some embodiments, the first chimeric polypeptide of a multi- chain chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) disposed between the at least one additional target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) and the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) or the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) positioned between the linker domain (e.g., any of the exemplary linker domains described herein) and the first domain of the pair of affinity domains (e.g., any of the first domains described herein or any of the exemplary pairs of affinity domains described herein), directly abuts the linker domain and/or the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide of a multi-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) disposed (i) between the linker domain (e.g., any of the exemplary linker domains described herein) and the at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) positioned between the linker domain (e.g., any of the exemplary linker domains described herein) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein), and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) at the N- terminal end and/or the C-terminal end of the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) directly abuts the second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) in the second chimeric polypeptide of a multi-chain chimeric polypeptide. In some

embodiments, the second chimeric polypeptide of a multi-chain chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target- binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) and the second domain of the pair of affinity domains (e.g., any of the second domains described herein of any of the exemplary pairs of affinity domains described herein) in the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains (e.g., any of the exemplary target- binding domains described herein or known in the art) directly abuts the second target- binding domain (e.g., any of the target-binding domains described herein or known in the art) in the second chimeric polypeptide of a multi-chain chimeric polypeptide. In some embodiments, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target-binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and the second target- binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) in the second chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains include the same amino acid sequence. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same antigen. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same epitope. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each include the same amino acid sequence.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to different antigens. In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen binding domain. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen-binding domain (e.g., a scFv or a single-domain antibody).

Pairs of Affinity Domains

In some embodiments, a multi-chain chimeric polypeptide includes: 1) a first chimeric polypeptide that includes a first domain of a pair of affinity domains, and 2) a second chimeric polypeptide that includes a second domain of a pair of affinity domains such that the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains. In some embodiments, the pair of affinity domains is a sushi domain from an alpha chain of an IL-15 receptor (ILl5Ra) (e.g., a human IL-15 receptor (IL-l5Ra)) and a soluble IL-15 (e.g., a human soluble IL-15). A sushi domain, also known as a short consensus repeat or type 1 glycoprotein motif, is a common motif in protein-protein interaction. Sushi domains have been identified on a number of protein-binding molecules, including complement components Clr, Cls, factor H, and C2m, as well as the nonimmunologic molecules factor XIII and P2-glycoprotein. Atypical Sushi domain has approximately 60 amino acid residues and contains four cysteines (Ranganathan, Pac. Symp Biocomput. 2000: 155-67). The first cysteine can form a disulfide bond with the third cysteine, and the second cysteine can form a disulfide bridge with the fourth cysteine. In some embodiments in which one member of the pair of affinity domains is a soluble IL-15, the soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments in which one member of the pair of affinity domains is an alpha chain of human IL-15 receptor (ILl5Ra), the human ILl5Ra is a mature full-length ILl5Ra. In some embodiments, the pair of affinity domains is barnase and barnstar. In some embodiments, the pair of affinity domains is a PKA and an AKAP. In some

embodiments, the pair of affinity domains is an adapter/docking tag module based on mutated RNase I fragments (Rossi, Proc Natl Acad Sci USA. 103:6841-6846, 2006;

Sharkey et ah, Cancer Res. 68:5282-5290, 2008; Rossi et ah, Trends Pharmacol Sci. 33:474-481, 2012) or SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25 (Deyev et ah, Nat Biotechnol . 1486-1492, 2003).

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide includes a first domain of a pair of affinity domains and a second chimeric polypeptide of the multi-chain chimeric polypeptide includes a second domain of a pair of affinity domains, wherein the first domain of the pair of affinity domains and the second domain of the pair of affinity domains bind to each other with a dissociation equilibrium constant (KD) of less than 1 x 10 ⁷ M, less than 1 x 10 ⁸ M, less than 1 x 10 ⁹ M, less than 1 x 10 ¹⁰ M, less than 1 x 10 ¹¹ M, less than 1 x 10 ¹² M, or less than 1 x 10 ¹³

M. In some embodiments, the first domain of the pair of affinity domains and the second domain of the pair of affinity domains bind to each other with a KD of about 1 x 10 ⁴ M to about 1 x 10 ⁶ M, about 1 x 10 ⁵ M to about 1 x 10 ⁷ M, about 1 x 10 ⁶ M to about 1 x 10 ⁸ M, about 1 x 10 ⁷ M to about 1 x 10 ⁹ M, about 1 x 10 ⁸ M to about 1 x 10 ¹⁰ M, about 1 x 10 ⁹ M to about 1 x 10 ¹¹ M, about 1 x 10 ¹⁰ M to about 1 x 10 ¹² M, about 1 x 10 ¹¹ M to about 1 x 10 ¹³ M, about 1 x 10 ⁴ M to about 1 x 10 ⁵ M, about 1 x 10 ⁵ M to about 1 x 10 ⁶ M, about 1 x 10 ⁶ M to about 1 x 10 ⁷ M, about 1 x 10 ⁷ M to about 1 x 10 ⁸ M, about 1 x 10 ⁸ M to about 1 x 10 ⁹ M, about 1 x 10 ⁹ M to about 1 x 10 ¹⁰ M, about 1 x 10 ¹⁰ M to about 1 x 10 ¹¹ M, about 1 x 10 ¹¹ M to about 1 x 10 ¹² M, or about 1 x 10 ¹² M to about 1 x 10 ¹³ M (inclusive). Any of a variety of different methods known in the art can be used to determine the KD value of the binding of the first domain of the pair of affinity domains and the second domain of the pair of affinity domains (e.g., an electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.).

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide includes a first domain of a pair of affinity domains and a second chimeric polypeptide of the multi-chain chimeric polypeptide includes a second domain of a pair of affinity domains, wherein the first domain of the pair of affinity domains, the second domain of the pair of affinity domains, or both is about 10 to 100 amino acids in length. For example, a first domain of a pair of affinity domains, a second domain of a pair of affinity domains, or both can be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino acids in length, about 10 to 85 amino acids in length, about 10 to 80 amino acids in length, about 10 to 75 amino acids in length, about 10 to 70 amino acids in length, about 10 to 65 amino acids in length, about 10 to 60 amino acids in length, about 10 to 55 amino acids in length, about 10 to 50 amino acids in length, about 10 to 45 amino acids in length, about 10 to 40 amino acids in length, about 10 to 35 amino acids in length, about 10 to 30 amino acids in length, about 10 to 25 amino acids in length, about 10 to 20 amino acids in length, about 10 to 15 amino acids in length, about 20 to 30 amino acids in length, about 30 to 40 amino acids in length, about 40 to 50 amino acids in length, about 50 to 60 amino acids in length, about 60 to 70 amino acids in length, about 70 to 80 amino acids in length, about 80 to 90 amino acids in length, about 90 to 100 amino acids in length, about 20 to 90 amino acids in length, about 30 to 80 amino acids in length, about 40 to 70 amino acids in length, about 50 to 60 amino acids in length, or any range in between. In some embodiments, a first domain of a pair of affinity domains, a second domain of a pair of affinity domains, or both is about 10, 15, 20, 25, 30, 35, 40, 45, 50,

55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

In some embodiments, any of the first and/or second domains of a pair of affinity domains disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at its N-terminus and/or C-terminus, so long as the function of the first and/or second domains of a pair of affinity domains remains intact. For example, a sushi domain from an alpha chain of human IL-15 receptor (ILl5Ra) can include one or more additional amino acids at the N-terminus and/or the C-terminus, while still retaining the ability to bind to a soluble IL-15. Additionally or alternatively, a soluble IL-15 can include one or more additional amino acids at the N-terminus and/or the C-terminus, while still retaining the ability to bind to a sushi domain from an alpha chain of human IL-15 receptor (ILl5Ra).

A non-limiting example of a sushi domain from an alpha chain of IL-15 receptor alpha (ILl5Ra) can include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to

ITCPPPMS VEHADIWVKS Y SLY SRERYICNSGFKRKAGTS SLTECVLNKATNVAH WTTPSLKCIR (SEQ ID NO: 10). In some embodiments, a sushi domain from an alpha chain of ILl5Ra can be encoded by a nucleic acid including

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAG CTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGA AGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGT GGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 11).

In some embodiments, a soluble IL-15 can include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to NW VN VI SDLKKIEDLIQ SMHID ATLYTE SD VHP SCK VTAMKCFLLELQ VI SLE SGD ASIHDTVENLIILANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV QMFINT

S (SEQ ID NO: 39). In some embodiments, a soluble IL-15 can be encoded by a nucleic acid including the sequence of

AACTGGGT GAACGT C ATC AGCGATTTAAAGAAGATCGAAGATTTAATTC AGTC CATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAA GGT GACCGCC AT GAAAT GTTTTTTACTGGAGCTGC AAGTTATCTCTTTAGAGAG CGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATA ACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGA AGAGCTGGAGGAGAAGAAC ATC AAGGAGTTTCTGCAATCCTTTGTGCACATTG TCCAGATGTTCATCAATACCTCC (SEQ ID NO: 110).

Signal Sequence

In some embodiments, a single-chain chimeric polypeptide includes a signal sequence at its N-terminal end. As will be understood by those of ordinary skill in the art, a signal sequence is an amino acid sequence that is present at the N-terminus of a number of endogenously produced proteins that directs the protein to the secretory pathway (e.g., the protein is directed to reside in certain intracellular organelles, to reside in the cell membrane, or to be secreted from the cell). Signal sequences are

heterogeneous and differ greatly in their primary amino acid sequences. However, signal sequences are typically 16 to 30 amino acids in length and include a hydrophilic, usually positively charged N-terminal region, a central hydrophobic domain, and a C-terminal region that contains the cleavage site for signal peptidase.

In some embodiments, a single-chain chimeric polypeptide includes a signal sequence having an amino acid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO: 62). In some embodiments, a single chain chimeric polypeptide includes a signal sequence encoded by the nucleic acid sequence

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC

(SEQ ID NO: 63),

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC

(SEQ ID NO: 64), or ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 65).

In some embodiments, a single-chain chimeric polypeptide includes a signal sequence having an amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 66). In some embodiments, a single-chain chimeric polypeptide includes a signal sequence having an amino acid sequence

MGQTVTMFE AT /PETTTDEVTNTVTTVT JTTTSTK AVYNEATCGTE AT VSF1 F1 AGR SC.G (SEQ ID NO: 67). In some embodiments, a single-chain chimeric polypeptide includes a signal sequence having an amino acid sequence

MPNHQSGSPTGSSDLLLSGKKQRPHLALRRKRRREMRKINRKVRRMNLAPIKEK TAWQHLQALISEAEEVLKTSQTPQNSLTLFLALLSVLGPPVTG (SEQ ID NO: 68).

In some embodiments, a single-chain chimeric polypeptide includes a signal sequence having an amino acid sequence MD SKGS SQKGSRLLLLLVV SNLLLCQGVV S (SEQ ID NO: 69). Those of ordinary skill in the art will be aware of other appropriate signal sequences for use in a single-chain chimeric polypeptide.

In some embodiments, a single-chain chimeric polypeptide includes a signal sequence that is about 10 to 100 amino acids in length. For example, a signal sequence can be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino acids in length, about 10 to 85 amino acids in length, about 10 to 80 amino acids in length, about 10 to 75 amino acids in length, about 10 to 70 amino acids in length, about 10 to 65 amino acids in length, about 10 to 60 amino acids in length, about 10 to 55 amino acids in length, about 10 to 50 amino acids in length, about 10 to 45 amino acids in length, about 10 to 40 amino acids in length, about 10 to 35 amino acids in length, about 10 to 30 amino acids in length, about 10 to 25 amino acids in length, about 10 to 20 amino acids in length, about 10 to 15 amino acids in length, about 20 to 30 amino acids in length, about 30 to 40 amino acids in length, about 40 to 50 amino acids in length, about 50 to 60 amino acids in length, about 60 to 70 amino acids in length, about 70 to 80 amino acids in length, about 80 to 90 amino acids in length, about 90 to 100 amino acids in length, about 20 to 90 amino acids in length, about 30 to 80 amino acids in length, about 40 to 70 amino acids in length, about 50 to 60 amino acids in length, or any range in between. In some embodiments, a signal sequence is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

In some embodiments, any of the signal sequences disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at its N-terminus and/or C-terminus, so long as the function of the signal sequence remains intact. For example, a signal sequence having the amino acid sequence

MKWVTFISLLFLFSSAYS (SEQ ID NO: 62) can include one or more additional amino acids at the N-terminus or C-terminus, while still retaining the ability to direct the single- chain chimeric polypeptide to the secretory pathway.

In some embodiments, a single-chain chimeric polypeptide includes a signal sequence that directs the single-chain chimeric polypeptide into the extracellular space. Such embodiments are useful in producing single-chain chimeric polypeptides that are relatively easy to be isolated and/or purified.

In some embodiments, a multi-chain chimeric polypeptide includes a first chimeric polypeptide that includes a signal sequence at its N-terminal end. In some embodiments, a multi-chain chimeric polypeptide includes a second chimeric polypeptide that includes a signal sequence at its N-terminal end. In some embodiments, both the first chimeric polypeptide of a multi-chain chimeric polypeptide and a second chimeric polypeptide of the multi-chain chimeric polypeptide include a signal sequence. As will be understood by those of ordinary skill in the art, a signal sequence is an amino acid sequence that is present at the N-terminus of a number of endogenously produced proteins that directs the protein to the secretory pathway (e.g., the protein is directed to reside in certain intracellular organelles, to reside in the cell membrane, or to be secreted from the cell). Signal sequences are heterogeneous and differ greatly in their primary amino acid sequences. However, signal sequences are typically 16 to 30 amino acids in length and include a hydrophilic, usually positively charged N-terminal region, a central hydrophobic domain, and a C-terminal region that contains the cleavage site for signal peptidase.

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence having an amino acid sequence

MKWVTFISLLFLFSSAYS (SEQ ID NO: 62). In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence encoded by the nucleic acid sequence

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC

(SEQ ID NO: 63),

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC

(SEQ ID NO: 64), or

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC

(SEQ ID NO: 65).

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence having an amino acid sequence

MKCLLYLAFLFLGVNC (SEQ ID NO: 66). In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence having an amino acid sequence

MGOTVTMFE AT /PHTTDEVTNTVTTVT JTTTSTK AVYNFATCGTE AT VSF1 F1 AGR SC.G

(SEQ ID NO: 67). In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence having an amino acid sequence

MPNHQSGSPTGSSDLLLSGKKQRPHLALRRKRRREMRKINRKVRRMNLAPIKEK TAWQHLQALISEAEEVLKTSQTPQNSLTLFLALLSVLGPPVTG (SEQ ID NO: 68).

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence having an amino acid sequence

MD SKGS SQKGSRLLLLLVV SNLLLCQGVV S (SEQ ID NO: 69). Those of ordinary skill in the art will be aware of other appropriate signal sequences for use in a first chimeric polypeptide and/or a second chimeric polypeptide of multi-chain chimeric polypeptides described herein.

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence that is about 10 to 100 amino acids in length. For example, a signal sequence can be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino acids in length, about 10 to 85 amino acids in length, about 10 to 80 amino acids in length, about 10 to 75 amino acids in length, about 10 to 70 amino acids in length, about

10 to 65 amino acids in length, about 10 to 60 amino acids in length, about 10 to 55 amino acids in length, about 10 to 50 amino acids in length, about 10 to 45 amino acids in length, about 10 to 40 amino acids in length, about 10 to 35 amino acids in length, about 10 to 30 amino acids in length, about 10 to 25 amino acids in length, about 10 to 20 amino acids in length, about 10 to 15 amino acids in length, about 20 to 30 amino acids in length, about 30 to 40 amino acids in length, about 40 to 50 amino acids in length, about 50 to 60 amino acids in length, about 60 to 70 amino acids in length, about 70 to 80 amino acids in length, about 80 to 90 amino acids in length, about 90 to 100 amino acids in length, about 20 to 90 amino acids in length, about 30 to 80 amino acids in length, about 40 to 70 amino acids in length, about 50 to 60 amino acids in length, or any range in between. In some embodiments, a signal sequence is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

In some embodiments, any of the signal sequences disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at its N-terminus and/or C-terminus, so long as the function of the signal sequence remains intact. For example, a signal sequence having the amino acid sequence

MKCLLYLAFLFLGVNC (SEQ ID NO: 66) can include one or more additional amino acids at the N-terminus or C-terminus, while still retaining the ability to direct a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both to the secretory pathway.

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence that directs the multi-chain chimeric polypeptide into the extracellular space. Such embodiments are useful in producing multi-chain chimeric polypeptides that are relatively easy to be isolated and/or purified.

Peptide Tags

In some embodiments, a single-chain chimeric polypeptide includes a peptide tag (e.g., at the N-terminal end or the C-terminal end of the single-chain chimeric

polypeptide). In some embodiments, a single-chain chimeric polypeptide includes two or more peptide tags.

Exemplary peptide tags that can be included in a single-chain chimeric polypeptide include, without limitation, AviTag (GLNDIFEAQKIEWHE; SEQ ID NO: 70), a calmodulin-tag (KRRWKKNFIAVS AANRFKKIS S SGAL; SEQ ID NO: 71), a polyglutamate tag (EEEEEE; SEQ ID NO: 72), an E-tag (GAPVPYPDPLEPR; SEQ ID NO: 73), a FLAG-tag (DYKDDDDK; SEQ ID NO: 74), an HA-tag, a peptide from hemagglutinin (YPYDVPDYA; SEQ ID NO: 75), a his-tag (HHHHH (SEQ ID NO: 76); HHHHHH (SEQ ID NO: 77); HHHHHHH (SEQ ID NO: 78); HHHHHHHH (SEQ ID NO: 79); HHHHHHHHH (SEQ ID NO: 80); or HHHHHHHHHH (SEQ ID NO: 81)), a myc-tag (EQKLISEEDL; SEQ ID NO: 82), NE-tag (TKENPRSNQEESYDDNES; SEQ ID NO: 83), S-tag (KETA AAKFERQHMD S ; SEQ ID NO: 84), SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP; SEQ ID NO: 85), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 86), Softag 3 (TQDPSRVG; SEQ ID NO: 87), Spot-tag (PDRVRAVSHWSS; SEQ ID NO: 88), Strep-tag (WSHPQFEK; SEQ ID NO: 89), TC tag (CCPGCC; SEQ ID NO: 90), Ty tag (EVHTNQDPLD; SEQ ID NO: 91), V5 tag (GKPIPNPLLGLD ST; SEQ ID NO: 92), VSV-tag (YTDIEMNRLGK; SEQ

ID NO: 93), and Xpress tag (DLYDDDDK; SEQ ID NO: 94). In some embodiments, tissue factor protein is a peptide tag.

Peptide tags that can be included in a single-chain chimeric polypeptide can be used in any of a variety of applications related to the single-chain chimeric polypeptide. For example, a peptide tag can be used in the purification of a single-chain chimeric polypeptide. As one non-limiting example, a single-chain chimeric polypeptide can include a myc tag; and can be purified using an antibody that recognizes the myc tag(s). One non-limiting example of an antibody that recognizes a myc tag is 9E10, available from the non-commercial Developmental Studies Hybridoma Bank. As another non- limiting example, a single-chain chimeric polypeptide can include a histidine tag, and can be purified using a nickel or cobalt chelate. Those of ordinary skill in the art will be aware of other suitable tags and agent that bind those tags for use in purifying a single chain chimeric polypeptide. In some embodiments, a peptide tag is removed from the single-chain chimeric polypeptide after purification. In some embodiments, a peptide tag is not removed from the single-chain chimeric polypeptide after purification.

Peptide tags that can be included in a single-chain chimeric polypeptide can be used, for example, in immunoprecipitation of the single-chain chimeric polypeptide, imaging of the single-chain chimeric polypeptide (e.g., via Western blotting, ELISA, flow cytometry, and/or immunocytochemistry), and/or solubilization of the single-chain chimeric polypeptide.

In some embodiments, a single-chain chimeric polypeptide includes a peptide tag that is about 10 to 100 amino acids in length. For example, a peptide tag can be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino acids in length, about 10 to 85 amino acids in length, about 10 to 80 amino acids in length, about 10 to 75 amino acids in length, about 10 to 70 amino acids in length, about 10 to 65 amino acids in length, about 10 to 60 amino acids in length, about 10 to 55 amino acids in length, about 10 to 50 amino acids in length, about 10 to 45 amino acids in length, about 10 to 40 amino acids in length, about 10 to 35 amino acids in length, about 10 to 30 amino acids in length, about 10 to 25 amino acids in length, about 10 to 20 amino acids in length, about 10 to 15 amino acids in length, about 20 to 30 amino acids in length, about 30 to 40 amino acids in length, about 40 to 50 amino acids in length, about 50 to 60 amino acids in length, about 60 to 70 amino acids in length, about 70 to 80 amino acids in length, about 80 to 90 amino acids in length, about 90 to 100 amino acids in length, about 20 to 90 amino acids in length, about 30 to 80 amino acids in length, about 40 to 70 amino acids in length, about 50 to 60 amino acids in length, or any range in between. In some embodiments, a peptide tag is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

Peptide tags included in a single-chain chimeric polypeptide can be of any suitable length. For example, peptide tags can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length. In embodiments in which a single-chain chimeric polypeptide includes two or more peptide tags, the two or more peptide tags can be of the same or different lengths. In some embodiments, any of the peptide tags disclosed herein may include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8,

9, 10, or more amino acids) at the N-terminus and/or C-terminus, so long as the function of the peptide tag remains intact. For example, a myc tag having the amino acid sequence EQKLISEEDL (SEQ ID NO: 82) can include one or more additional amino acids (e.g., at the N-terminus and/or the C- terminus of the peptide tag), while still retaining the ability to be bound by an antibody (e.g., 9E10). In some embodiments, a multi-chain chimeric polypeptide includes a first chimeric polypeptide that includes a peptide tag (e.g., at the N-terminal end or the C- terminal end of the first chimeric polypeptide). In some embodiments, a multi-chain chimeric polypeptide includes a second chimeric polypeptide that includes a peptide tag (e.g., at the N-terminal end or the C-terminal end of the second chimeric polypeptide). In some embodiments, both the first chimeric polypeptide of a multi-chain chimeric polypeptide and a second chimeric polypeptide of the multi-chain chimeric polypeptide include a peptide tag. In some embodiments, a first chimeric polypeptide of a multi- chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both include two or more peptide tags.

Exemplary peptide tags that can be included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both include, without limitation, AviTag

(GLNDIFEAQKIEWHE; SEQ ID NO: 70), a calmodulin-tag

(KRRWKKNFIAVS AANRFKKIS SSGAL; SEQ ID NO : 71 ), a polyglutamate tag

(EEEEEE; SEQ ID NO: 72), an E-tag (GAPVPYPDPLEPR; SEQ ID NO: 73), a FLAG- tag (DYKDDDDK; SEQ ID NO: 74), an HA-tag, a peptide from hemagglutinin

(YPYDVPDYA; SEQ ID NO: 75), a his-tag (HHHHH (SEQ ID NO: 76); HHHHHH (SEQ ID NO: 77); HHHHHHH (SEQ ID NO: 78); HHHHHHHH (SEQ ID NO: 79); HHHHHHHHH (SEQ ID NO: 80); or HHHHHHHHHH (SEQ ID NO: 81), a myc-tag (EQKLISEEDL; SEQ ID NO: 82), NE-tag (TKENPRSN QEES YDDNES ; SEQ ID NO: 83), S-tag, (KETA AAKFERQHMD S ; SEQ ID NO: 84), SBP-tag

(MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP; SEQ ID NO: 85), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 86), Softag 3 (TQDPSRVG; SEQ ID NO: 87), Spot-tag (PDRVRAVSHWSS; SEQ ID NO: 88), Strep-tag (WSHPQFEK; SEQ ID

NO: 89), TC tag (CCPGCC; SEQ ID NO: 90), Ty tag (EVHTNQDPLD; SEQ ID NO:

91), V5 tag (GKPIPNPLLGLD ST; SEQ ID NO: 92), VSV-tag (YTDIEMNRLGK; SEQ ID NO: 93), and Xpress tag (DLYDDDDK; SEQ ID NO: 94). In some embodiments, tissue factor protein is a peptide tag.

Peptide tags that can be included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both can be used in any of a variety of applications related to the multi- chain chimeric polypeptide. For example, a peptide tag can be used in the purification of a multi-chain chimeric polypeptide. As one non-limiting example, a first chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed first chimeric polypeptide), a second chimeric polypeptide of the multi-chain chimeric polypeptide (e.g., a recombinantly expressed second chimeric polypeptide), or both can include a myc tag; the multi-chain chimeric polypeptide that includes the myc-tagged first chimeric polypeptide, the myc-tagged second chimeric polypeptide, or both can be purified using an antibody that recognizes the myc tag(s). One non-limiting example of an antibody that recognizes a myc tag is 9E10, available from the non-commercial Developmental Studies Hybridoma Bank. As another non-limiting example, a first chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed first chimeric polypeptide), a second chimeric polypeptide of the multi-chain chimeric polypeptide (e.g., a recombinantly expressed second chimeric polypeptide), or both can include a histidine tag; the multi-chain chimeric polypeptide that includes the histidine-tagged first chimeric polypeptide, the histidine-tagged second chimeric polypeptide, or both can be purified using a nickel or cobalt chelate. Those of ordinary skill in the art will be aware of other suitable tags and agent that bind those tags for use in purifying multi-chain chimeric polypeptide. In some embodiments, a peptide tag is removed from the first chimeric polypeptide and/or the second chimeric polypeptide of the multi-chain chimeric polypeptide after purification. In some embodiments, a peptide tag is not removed from the first chimeric polypeptide and/or the second chimeric polypeptide of the multi-chain chimeric polypeptide after purification.

Peptide tags that can be included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both can be used, for example, in immunoprecipitation of the multi-chain chimeric polypeptide, imaging of the multi-chain chimeric polypeptide (e.g., via Western blotting, ELISA, flow cytometry, and/or immunocytochemistry), and/or solubilization of the multi-chain chimeric polypeptide.

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a peptide tag that is about 10 to 100 amino acids in length. For example, a peptide tag can be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino acids in length, about 10 to 85 amino acids in length, about 10 to 80 amino acids in length, about 10 to 75 amino acids in length, about 10 to 70 amino acids in length, about 10 to 65 amino acids in length, about 10 to 60 amino acids in length, about 10 to 55 amino acids in length, about 10 to 50 amino acids in length, about 10 to 45 amino acids in length, about 10 to 40 amino acids in length, about 10 to 35 amino acids in length, about 10 to 30 amino acids in length, about 10 to 25 amino acids in length, about 10 to 20 amino acids in length, about 10 to 15 amino acids in length, about 20 to 30 amino acids in length, about 30 to 40 amino acids in length, about 40 to 50 amino acids in length, about 50 to 60 amino acids in length, about 60 to 70 amino acids in length, about 70 to 80 amino acids in length, about 80 to 90 amino acids in length, about 90 to 100 amino acids in length, about 20 to 90 amino acids in length, about 30 to 80 amino acids in length, about 40 to 70 amino acids in length, about 50 to 60 amino acids in length, or any range in between. In some embodiments, a peptide tag is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,

70, 75, 80, 85, 90, 95, or 100 amino acids in length.

Peptide tags included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both can be of any suitable length. For example, peptide tags can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length. In embodiments in which a multi-chain chimeric polypeptide includes two or more peptide tags, the two or more peptide tags can be of the same or different lengths. In some embodiments, any of the peptide tags disclosed herein may include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at the N-terminus and/or C-terminus, so long as the function of the peptide tag remains intact. For example, a myc tag having the amino acid sequence EQKLISEEDL (SEQ ID NO: 82) can include one or more additional amino acids (e.g., at the N-terminus and/or the C- terminus of the peptide tag), while still retaining the ability to be bound by an antibody (e.g., 9E10).

Exemplary Embodiments of Single-Chain Chimeric Polypeptides- Type A

In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target-binding domain and/or the second target-binding domain can independently bind specifically to CD3 (e.g., human CD3) or CD28 (e.g., human CD28). In some embodiments, the first target-binding domain binds specifically to CD3 (e.g., human CD3) and the second target-binding domain binds specifically to CD28 (e.g., human CD28). In some embodiments, the first target-binding domain binds specifically to CD28 (e.g., human CD28) and the second target-binding domain binds specifically to CD3 (e.g., human CD3).

In some embodiments of these single-chain chimeric polypeptides, the first target- binding domain and the soluble tissue factor domain directly abut each other. In some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain.

In some embodiments of these single-chain chimeric polypeptides, the soluble tissue factor domain and the second target-binding domain directly abut each other. In some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the second target-binding domain.

In some embodiments of these single-chain chimeric polypeptides, one or both of the first target-binding domain and the second target-binding domain is an antigen binding domain. In some embodiments of these single-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain are each an antigen- binding domain (e.g., any of the exemplary antigen-binding domains described herein).

In some embodiments of these single-chain chimeric polypeptides, the antigen-binding domain includes a scFv or a single domain antibody.

A non-limiting example of an scFv that binds specifically to CD3 can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QIVLT Q SP AIMS ASPGEK VTMT C S AS S S VS YMNW Y QQK SGTSPKRWIYDT SKL A S GVP AHFRGS GS GT S Y SLTI S GME AED A AT Y Y C QQ W S SNPFTF GS GTKLEINRGG GGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQ RPGQGLEWIGYINP SRGYTNYN QKFKDK ATLTTDK S S S T A YMQL S SLT SED S A V YYCARYYDDHYCLDYWGQGTTLTVSS (SEQ ID NO: 20).

In some embodiments, an scFv that binds specifically to CD3 can be encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGA

AGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTAT

CAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAG

CTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTA

CTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCC

AGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAAT

CAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAG

CCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCC

GTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCA

TTGGGT C AAGC AGAGGCCCGGT C AAGGTTT AGAGT GGATCGGAT AT ATC AAC

CCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTT

TAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACC

AGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTG TTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGC (SEQ ID NO: 12).

A non-limiting example of an scFv that binds specifically to CD28 can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYN D YTK YNEKFKGK ATLT SDKS SIT AYMEF S SLT SED S AL Y Y C ARW GDGNYW GRG TTLT VS SGGGGSGGGGSGGGGSDIEMTQ SP AIMS ASLGERVTMTCT AS S S VS S S Y FHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCH Q YHRSPTF GGGTKLETKR (SEQ ID NO: 26).

In some embodiments, an scFv that binds specifically to CD28 can be encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGA AAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGG GTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTT ACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAAC CTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCG AGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGGG ACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGAGGCTCCGGCGGAGG CGGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTATC ATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAG CGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTA AACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTT TTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCT GAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGG AGGC GGC AC C A A AC T GGAGAC A A AG AGG (SEQ ID NO: 13).

In some embodiments of these single-chain chimeric polypeptides, the first target- binding domain and/or the second target-binding domain is a soluble receptor (e.g., a soluble CD28 receptor or a soluble CD3 receptor). In some embodiments of these single- chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein.

In some embodiments, a single-chain chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QIVLT Q SP AIMS ASPGEK VTMT C S AS S S VS YMNW Y QQK SGTSPKRWIYDT SKL A S GVP AHFRGS GS GT S Y SLTI S GME AED A AT Y Y C QQ W S SNPFTF GS GTKLEINRGG GGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQ RPGQGLEWIGYINP SRGYTNYN QKFKDK ATLTTDK S S S T A YMQL S SLT SED S A V YY C ARYYDDHY CLD YW GQGTTLT V S S SGTTNT VAAYNLTWKSTNFKTILEWEP KPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAG NVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRR NNTFL SLRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNEFLID VDKGEN Y CF S VQ AVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKASG YTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAY MEF S SLTSEDS ALYYC ARWGDGNYWGRGTTLT VS SGGGGSGGGGSGGGGSDIE MTQ SP AIMS ASLGERVTMTCT AS S S VS S S YFHW YQQKPGS SPKLCI YSTSNL ASG VPPRF SGSGST S Y SLTIS SME AED AATYF CHQ YHRSPTF GGGTKLETKR (SEQ ID NO: 100).

In some embodiments, a single-chain chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGA

AGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTAT

CAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAG

CTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTA

CTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCC

AGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAAT

CAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAG

CCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCC

GTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCA

TTGGGT C AAGC AGAGGCCCGGT C AAGGTTT AGAGT GGATCGGAT AT ATC AAC

CCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTT

TAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACC

AGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTG

TTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCACC

ACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGA

CAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGAT

CTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACC

GAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGG

CTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGC

GAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTT

AGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTC

ACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCC

TCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCC

AGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTG

ACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTC

TCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAA

GAAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTC

GTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCT

TCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGA GTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAG

TTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACA

TGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGG

TGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGC

GGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATC

GAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCA

CAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTAC

CAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATC

TCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTC

TTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACC

AGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGA

GG (SEQ ID NO: 101).

In some embodiments, a single-chain chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQ KSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWS SNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCK ASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSS S T A YMQL S SLT SED S A V Y Y C ARY YDDH Y CLD YW GQGTTLT V S S S GTTNT V A AY NLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVK DVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVG TK VN VT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNE FLID VDKGEN Y CF S VQ A VIP SRT VNRK S TD SP VECMGQEKGEFRE V QLQ Q S GPEL VKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKF KGK ATLT SDK S SIT A YMEF S SLT SED S AL Y Y C ARW GDGNYW GRGTTLT V S S GGG GSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPG S SPKLCI YSTSNL ASGVPPRF SGSGST S Y SLTIS SME AED AAT YF CHQ YHRSPTF GG GTKLETKR (SEQ ID NO: 102). In some embodiments, a single-chain chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTTATTCAGCTCCGCCTATTCC

CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGA

AGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTAT

CAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAG

CTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTA

CTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCC

AGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAAT

CAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAG

CCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCC

GTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCA

TTGGGT C AAGC AGAGGCCCGGT C AAGGTTT AGAGT GGATCGGAT AT ATC AAC

CCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTT

TAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACC

AGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTG

TTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCACC

ACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGA

CAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGAT

CTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACC

GAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGG

CTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGC

GAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTT

AGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTC

ACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCC

TCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCC

AGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTG

ACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTC TCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAA GAAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTC GTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCT TCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGA GTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAG TTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACA TGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGG TGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGC GGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATC GAGATGACCCAGTCCCCCGCTATC ATGTCCGCCTCTTTAGGCGAGCGGGTC A CAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTAC CAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATC TCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTC TTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACC AGTACCACCGGTCCCCC ACCTTCGGAGGCGGC ACC AAACTGGAGAC AAAGA GG (SEQ ID NO: 103).

Exemplary Embodiments of Single-Chain Chimeric Polypeptides- Type B

In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target-binding domain and/or the second target-binding domain can independently bind specifically to an IL-2 receptor (e.g., human IL-2 receptor).

In some embodiments of these single-chain chimeric polypeptides, the first target- binding domain and the soluble tissue factor domain directly abut each other. In some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain.

In some embodiments of these single-chain chimeric polypeptides, the soluble tissue factor domain and the second target-binding domain directly abut each other. In some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the second target-binding domain.

In some embodiments of these single-chain chimeric polypeptides, the first target- binding domain and the second target-binding domain is a soluble human IL-2 protein. A non-limiting example of an IL-2 protein that binds specifically to an IL-2 receptor can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELK

HLQCLEEELKPLEEVLNL AQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADE T ATIVEFLNRWITF CQ SIISTLT (SEQ ID NO: 28).

In some embodiments, an IL-2 protein that binds specifically to an IL-2 receptor can be encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTAC TGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAA ACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAA CTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGC TAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAG C A AT AT C A AC GT A AT AGTTCTGGA AC T A A AGGGATCTGA A AC A AC ATT C ATG T GT GA AT AT GCTGAT GAG AC AGC A ACC ATTGT AGA ATTTC T GA AC AG AT GGA TTACCTTTTGTCAAAGCATCATCTCAACACTAACT (SEQ ID NO: 104).

In some embodiments, an IL-2 protein that binds specifically to an IL-2 receptor can be encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTAC

TGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAA

GCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAG

CTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTGC

TGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGC

AACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGT

GCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGAT

CACCTTCTGCCAGTCCATCATCTCCACTTTAACC (SEQ ID NO: 105)

In some embodiments of these single-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein.

In some embodiments, a single-chain chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELK HLQCLEEELKPLEEVLNL AQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADE T ATI VEFLNRWITF C Q SII STLT S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTG S AGEPL YEN SPEFTP YLETNLGQPTIQ SFEQ VGTK VNVTVEDERTL VRRNNTFL SL RDVFGKDLIYTL YYWKS S S SGKKTAKTNTNEFLIDVDKGENYCF S VQ AVIPSRTV NRK S TD SP VECMGQEKGEFRE APT S S S TKKTQLQLEHLLLDLQMILN GINN YKNP KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNI N VI VLELKGSETTFMCE Y ADET ATI VEFLNRWITF C Q SII S TLT (SEQ ID NO: 106).

In some embodiments, a single-chain chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTAC

TGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAA

GCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAG

CTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTGC

TGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGC

AACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGT

GCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGAT

CACCTTCTGCCAGTCCATCATCTCCACTTTAACCAGCGGCACAACCAACACAG

TCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGA

ATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAG

TCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATC

TCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTT

TAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTA

TACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGC

CCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGA

GGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGAT

GTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTC

CGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGAT

AAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCG

T GA AT AGGA A A AGC ACC GAT AGCC CC GTT GAGT GC AT GGGC C A AGA A A AGG

GCGAGTTCCGGGAGGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACA

ACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATT

ACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAA

GAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCT

CTGGAGGAAGT GCT AAATTT AGCTC A AAGC AAAA ACTTT C ACTT AAGACCC A

GGG AC TT A AT C AGC A AT AT C A AC GT A AT AGT TC T GG A AC T A A AGGG AT C T G A

AAC AAC ATTC AT GT GT GAAT AT GCTGAT GAGAC AGC AACC ATTGT AGAATTT

CTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTAACT (SEQ

ID NO: 107).

In some embodiments, a single-chain chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFS SAYS APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTR MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVL ELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSGTTNTVAAYNLTWKST NFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTY LARVF S YPAGNVESTGS AGEPLYENSPEFTP YLETNLGQPTIQ SFEQ VGTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNEFLID VDK GENY CF S VQ AVIP SRT VNRK STD SP VECMGQEKGEFREAPT S S STKKT QLQLEHL LLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN L AQ SKNFHLRPRDLISNINVIVLELKGSETTFMCE Y ADET ATIVEFLNRWITF CQ SII STLT (SEQ ID NO: 108).

In some embodiments, a single-chain chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CGCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTA

CTGCTGGATTT AC AG AT GATTTT AAACGGC ATC A AC AACT AC A AGAACCCC A

AGCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGA

GCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTG

CTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAG

CAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATG

TGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGA

TCACCTTCTGCCAGTCCATCATCTCCACTTTAACCAGCGGCACAACCAACACA

GTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCG

AATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAA

GTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGAT T ACCTCGCCCGGGTGT TTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTT ATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAG CCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGG AGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGA TGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCT CCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGA TAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACC GT GA AT AGG A A A AGC AC CGAT AGC CCC GTT GAGT GC AT GGGC C A AGA A A AG GGCGAGTTCCGGGAGGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTAC AACTGGAGC ATTT ACTGCTGGATTTAC AGATGATTTTGAATGGAATT AAT AAT TACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAA GAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCT CTGGAGGAAGT GCT AAATTT AGCTC A AAGC AAAA ACTTT C ACTT AAGACCC A GGG AC TT AAT C AGC A AT AT C A AC GT AAT AGT TC T GG A AC T A A AGGG AT C T G A AAC AAC ATTC AT GT GT GAAT AT GCTGAT GAGAC AGC AACC ATTGT AGAATTT CTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTAACT (SEQ ID NO: 109).

Exemplary Embodiments of Single-Chain Chimeric Polypeptides- Type C

In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target-binding domain and/or the second target-binding domain can independently bind specifically to an IL-15 receptor (e.g., a human IL-15 receptor).

In some embodiments of these single-chain chimeric polypeptides, the first target- binding domain and the soluble tissue factor domain directly abut each other. In some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain.

In some embodiments of these single-chain chimeric polypeptides, the soluble tissue factor domain and the second target-binding domain directly abut each other. In some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the second target-binding domain.

In some embodiments of these single-chain chimeric polypeptides, the first target- binding domain and the second target-binding domain is a soluble human IL-15 protein.

A non-limiting example of an IL-15 protein that binds specifically to an IL-15 receptor can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI SLE SG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS (SEQ ID NO: 39).

In some embodiments, an IL-15 protein that binds specifically to an IL-15 receptor can be encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

AACTGGGT GAACGT GAT C AGCGATTT AAAGAAGATCGAGGATTT AATCC AGA GCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTAGCTG CAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTAG AGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTTTAGC C AAC AACTCTTT AAGC AGC AACGGC AACGT GAC AGAGAGCGGCTGC AAGGA GT GCGAGGAGCTGGAGGAGA AGAAC AT C AAGGAGTTTTT AC AGAGCTTCGT G C AC ATCGTGC AGAT GTT CAT C AAC ACT AGC (SEQ ID NO: 111).

In some embodiments, an IL-15 protein that binds specifically to an IL-15 receptor can be encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: AACTGGGT GAACGT CAT C AGCGATTT AAAGAAGATCGA AGATTT A ATT C AGT CCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGT AAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGA GAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCC AATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGT GCGA AGAGCTGGAGGAGAAGAAC ATC AAGGAGTTTCTGC AATCCTTT GT GCA CATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 110)

In some embodiments of these single-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein.

In some embodiments, a single-chain chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI SLE SG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN T S S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QI S TK S GD WK SKCF YT TDTECDLTDEIVKD VKQTYLARVF S YP AGNVESTGS AGEPLYEN SPEFTPYLETN LGQPTIQ SFEQ V GTK VN VT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNEFLID VDKGEN Y CF S VQ A VIP SRT VNRK S TD SP VECMGQEKGE FRENW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VISLE SGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV QMF INTS (SEQ ID NO: 112). In some embodiments, a single-chain chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: AACTGGGT GAACGT GAT C AGCGATTT AAAGAAGATCGAGGATTT AATCC AGA

GCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTAGCTG

CAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTAG

AGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTTTAGC

C AAC AACTCTTT AAGC AGC AACGGC AACGT GAC AGAGAGCGGCTGC AAGGA

GT GCGAGGAGCTGGAGGAGA AGAAC AT C AAGGAGTTTTT AC AGAGCTTCGT G

CACATCGTGCAGATGTTCATCAACACTAGCAGCGGCACAACCAACACAGTCG

CTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATG

GGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCC

GGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCA

CCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAG

CTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATAC

GAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCA

CCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGA

CGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTG

TTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGG

C AAGA AGAC AGCT AAAACC AAC AC AAACGAGTTTTT AATCGACGTGGAT AAA

GGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGA

ATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCG

AGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGA

TTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGC

ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTT

ATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAA

TCATTTTAGCCAATAACTCTTTATCC AGC AACGGC AACGTGACAGAGTCCGGC

T GC A AGGAGT GC GA AGAGCTGGAGGAGA AGA AC AT C A AGGAGTTTC T GCA A

TCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 113).

In some embodiments, a single-chain chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: MKWVTFISLLFLFSSAYSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVT AMKCFLLELQ VI SLE SGD A SIHDT VENLIIL ANN SL S SN GN VTE S GCKECEELEEK NIKEFLQ SF VHI V QMFINT S SGTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT VQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSA GEPL YEN SPEF TP YLETNLGQPTIQ SFEQ V GTK VNT VT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNEFLID VDKGEN Y CF S VQ A VIP SRT VNR K S TD SP VECMGQEKGEFRENW VN VI SDLKKIEDLIQ SMHID ATL YTESD VHP S CK VT AMKCFLLELQ VISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELE EKNIKEFLQ SF VHI V QMFINT S (SEQ ID NO: 1 14).

In some embodiments, a single-chain chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CAACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAG

AGCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTAGCT

GCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTA

GAGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTTTAG

CCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGG

AGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCGT

GCACATCGTGCAGATGTTCATCAACACTAGCAGCGGCACAACCAACACAGTC

GCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAAT

GGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTC

CGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTC

ACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTA

GCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATA

CGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCC

ACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGG

ACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGT

GTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCG GCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAA AGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTG A AT AGG A A A AGC AC C GAT AGC C C C GT T G AGT GC AT GGGC C A AG A A A AGGGC GAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAG ATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTG CACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGT TATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTA ATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCG GCTGC AAGGAGTGCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTGTGC AC ATTGTCC AGATGTTC ATC AAT ACCTCC (SEQ ID NO : 115).

Exemplary Multi-Chain Chimeric Polypeptides- Type A

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-18 or a receptor of IL-12. In some examples of these multi-chain chimeric polypeptides, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target-binding domain and the second target-binding domain is an agonistic antigen-binding domain. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain and the second target-binding domain are each agonistic antigen-binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target-binding domain and the second target-binding domain is a soluble IL-15 or a soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain are each independently a soluble IL-15 or a soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain both bind specifically to a receptor of IL-18 or a receptor of IL-12. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain binds specifically to a receptor for IL-12, and the second target-binding domain binds specifically to a receptor for IL-18. In some embodiments of these multi chain chimeric polypeptides, the first target-binding domain binds specifically to a receptor for IL-18, and the second target-binding domain bind specifically to a receptor for IL-12.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain includes a soluble IL-18 (e.g., a soluble human IL-18).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-18 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQ PRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKM QFES S S YEGYFL ACEKERDLFKLILKKEDELGDRSIMFT VQNED (SEQ ID NO : 41 ).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-18 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACC AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGAC TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA AATCAGC ACTTTATCTTGTGAGAACAAGATC ATCTCCTTTAAGGAAATGAACC CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT

(SEQ ID NO: 42).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain includes a soluble IL-12 (e.g., a soluble human IL-12). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-12 includes a sequence of soluble human IE-12b (p40) and a sequence of soluble human IL- 12a (p35). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-12 further includes a linker sequence (e.g., any of the exemplary linker sequences described herein) between the sequence of soluble IE-12b (p40) and the sequence of soluble human IL-l2a (p35). In some examples of these multi-chain chimeric polypeptides, the linker sequence comprises GGGGSGGGGSGGGGS (SEQ ID NO: 7). In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human P.-12b (p40) comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKEF GD AGQ YTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQGVTCGAATLS AERVRGDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKN LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT SATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 33).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IE-12b (p40) is encoded by a sequence that is at least 80% identical (e.g., at least

82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG

ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG

CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA

AGGGAGGCGAGGT GCTC AGCC ATTCCTT ATT ATT ATT AC AC AAGAAGGAAGA

CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG

ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG

GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA

GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG

ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC

GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA

CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA

CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGC T GGTCCGAGT GGGCC AGCGT GCCTT GTTCC (SEQ ID NO: 34).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-l2a (p35) includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKD KTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKM Y QVEFKTMNAKLLMDPKRQIFLDQNML AVIDELMQ ALNFN SETVPQKS SLEEPD FYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 35).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-l2a (p35) is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCA CAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAG ACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCA CCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAA GAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTT GTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCT ACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCT GCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTG ATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGA AGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCAT TTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCT ATTTAAACGCCAGC (SEQ ID NO: 36). In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQ PRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKM QFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYN LTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKD VKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGT K VN VT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNEF LID VDKGEN Y CF S VQ A VIP SRT VNRK S TD SP VECMGQEKGEFRENW VN VISDLK KIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLI IL ANN SL S SN GN VTE S GCKECEELEEKNIKEFLQ SF VHI V QMFINT S (SEQ ID NO: 116).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACC

AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGAC

TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA

GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA

AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACC

CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC

GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT

ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA

GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA

ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACC

ACCGAC ACCGAGT GCGATCTC ACCGAT GAGATCGT GAAAGAT GT GAAAC AGA

CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG

TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG

AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA

GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACAC

CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT

ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA

GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT

GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT

GC AT GGGCC AAGAA AAGGGCGAGTTCCGGGAGAACTGGGT GAACGT CAT C A

GCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCAC

TTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT

GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATC

CACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAA

CGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAA

GAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCA

ATACCTCC (SEQ ID NO: 1 17).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD CRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKD TKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM F TV QNED S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QI STK S GD WK S KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP YLETNLGQPTIQ SFEQ V GTKVNVTVEDERTLVRRNNTFL SLRD VF GKDLI YTL YY WKSS S SGKKTAKTNTNEFLIDVDKGENYCF S VQ A VIP SRT VNRK S TD SP VECMG

QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL Q VI SLE S GD ASIHD T VENLIIL ANN SL S SN GN VTE S GCKECEELEEKNIKEFLQ SF V HIVQMFINTS (SEQ ID NO: 1 18).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACC

AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGAC

TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA

GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA

AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACC

CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC

GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT

ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA

GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA

ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC

CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACC

ACCGAC ACCGAGT GCGATCTC ACCGAT GAGATCGT GAAAGAT GT GAAAC AGA

CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG

TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG

AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA

GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACAC

CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT

ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA

GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT

GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT

GC AT GGGCC AAGAA AAGGGCGAGTTCCGGGAGAACTGGGT GAACGT CAT C A

GCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCAC TTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT _{GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATC} CACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAA CGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAA GAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCA ATACCTCC (SEQ ID NO: 119).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLT

IQVKEF GD AGQ YTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKNKTFLR

CEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQGVTCGAATLS AERVRGDN

KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKN

LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT

SATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPV

ATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTV

EACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEF

KTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFN SETVPQKS SLEEPDFYKTK

IKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICN

S GFKRK AGT S SLTEC VLNKATN V AHWTTP SLKCIR (SEQ ID NO: 120).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG

ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG

CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA

AGGGAGGCGAGGT GCTC AGCC ATTCCTT ATT ATT ATT AC AC AAGAAGGAAGA

CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG

ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG

GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA

AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA

GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG

ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC

GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA

CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA

GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA

CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG

AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG

GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGC

TGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAG

GTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCC

CGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGC

AACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCG

AGGAGATCGACC AT GAAGAT AT C ACC AAGGAC AAGAC ATCC ACCGT GGAGG

CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAA

ACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTAT

GATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGG

AGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTT

TTT AGACC AGAAC AT GCTGGCTGT GATT GAT GAGCTGATGC AAGCTTT AAACT

TCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTAC

AAGAC AAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCG

TGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCT

CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA

GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA

GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC

AACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 121). In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKW VTFISLLFLF S SAY SIWELKKD VYVVELDW YPD APGEMVVLT CDTPEEDGI TWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQG VTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYE NYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQV QGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGG GSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYP CT SEEIDHEDITKDKT S T VE ACLPLELTKNE S CLN SRET SFITN GS CL ASRKT SFMM ALCLS SIYEDLKMY Q VEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQ ALNFN S ETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHA DIWVKS Y SLY SRERYICNSGFKRK AGT S SLTEC VLNK ATNVAHWTTP SLKCIR

(SEQ ID NO: 122).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG

ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG

CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC

CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA

AGGGAGGCGAGGT GCTC AGCC ATTCCTT ATT ATT ATT AC AC AAGAAGGAAGA

CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG

ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG

GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA

GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG

ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC

GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA

CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA

GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA

CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG

AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG

GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGC

TGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAG

GTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCC

CGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGC

AACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCG

AGGAGATCGACC AT GAAGAT AT C ACC AAGGAC AAGAC ATCC ACCGT GGAGG

CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAA

ACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTAT

GATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGG

AGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTT

TTT AGACC AGAAC AT GCTGGCTGT GATT GAT GAGCTGATGC AAGCTTT AAACT

TCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTAC

AAGAC AAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCG

TGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCT

CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA

GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA

GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC

AACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 123).

Exemplary Multi-Chain Chimeric Polypeptides- Type B

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-21 or to TGF-b. In some examples of these multi-chain chimeric polypeptides, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target-binding domain and the second target-binding domain is a soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or a soluble TGF-b receptor (e.g., a soluble TGF-b RII receptor). In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain are each

independently a soluble IL-21 or a soluble TGF-b receptor (e.g., a soluble TGF-b RII receptor). In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain both bind specifically to a receptor of IL-21 or to TGF-b. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain binds specifically to a receptor for IL-21, and the second target-binding domain binds specifically to TGF-b. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain binds specifically to TGF-b, and the second target-binding domain bind specifically to a receptor for IL-21.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain includes a soluble IL-21 (e.g., a soluble human IL-21). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKS ANTGNNERIINV SIKKLKRKPP STNAGRRQKHRLTCP SCD S YEKKPPKEFLER

FKSLLQKMIHQHL S SRTHGSED S (SEQ ID NO: 124).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG

ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC

CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG

AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 125).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain includes a soluble TGF-b receptor (e.g., a soluble TGF-b RII receptor (e.g., a soluble human TGF-b RII receptor)). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII includes a first sequence of soluble human TGF-b RII and a second sequence of soluble human TGF-b RII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII includes a linker disposed between the first sequence of soluble human TGF-b RII and the second sequence of soluble human TGF-b RII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence

GGGGS GGGGS GGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56). In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCA TCTGCGAGAAGCCCC AAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 57).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCA TCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGA GAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGAT TTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGA AGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGA CAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 57). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 61).

In some embodiments of these multi-chain chimeric polypeptides, the human TGFpRII receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM

IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIF SEE YNT SNPD (SEQ ID NO: 60).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKS ANTGNNERIINV SIKKLKRKPP STNAGRRQKHRLTCP SCD S YEKKPPKEFLER FK SLLQKMIHQHL S SRTHGSED S S GTTNT V A A YNLTWK S TNFKTILEWEPKP VN Q VYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVEST GSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLS LRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNEFLID VDKGEN Y CF S VQ A VIP SRT VNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHP SCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECE ELEEKNIKEFLQ SF VHIV QMFINTS (SEQ ID NO: 126).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG

ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC

CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG

GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG

AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG

CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC

CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA

GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCTCCGGCACCACCAAT

ACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTC TGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCAC

CAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGT

GATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGG

TCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCT

CTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCA

GCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTC

GAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGG

ATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCC

TCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGG

ACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTAC

CGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAA

GGGC G AGTT C C GGG AG A AC T GGGT G A AC GT CAT C AGC GAT T T A A AG A AG AT C

GAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGA

CGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGC

AAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAA

TTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGT

CCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTC

TGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO:

127).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEW SAF SCF QKAQLKS ANTGNNERIINV SIKKLKRKPPSTNAGRRQKHRLT CP S CD S YEKKPPKEFLERFK SLLQKMIHQHL S SRTHGSED S SGTTNT V A A YNLT W KSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQ T YL ARVF S YP AGNVE S T GS AGEPL YEN SPEF TP YLETNLGQPTIQ SFEQ V GTK VN VT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNEFLID V

DKGEN Y CF S VQ A VIP SRT VNRK S TD SP VECMGQEKGEFRENW VN VI SDLKKIED LIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAN NSL S SN GN VTES GCKECEELEEKNIKEFLQ SF VHI V QMFINT S (SEQ ID NO: 128).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC

GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC

CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA

GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGT

GAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAG

GCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCC

CCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATC

AGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCTCCGGCACCACCAA

TACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATT

CTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCA

CCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGT

GTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCG

GGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGC

CTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGC

CAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCG

TCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCG

GGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCT

CCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGT

GGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGT

ACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAA

AGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGAT

CGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCG ACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTG

CAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGA

ATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAG

TCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTT

CTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO:

129).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE

KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET

FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM

IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK

NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND

NIIF SEEYNTSNPDITCPPPMS VEHADIWVKS Y SLY SRERYICNSGFKRKAGTS SLT

ECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 130)

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA

ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC

AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCA

TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG

AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA

CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG

AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGACATCACGTGTCCTCCTCCTATGTCC GTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGC GGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGAC GGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGT CTCAAATGTATTAGA (SEQ ID NO: 131). In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S SAY SIPPHV QKS VNNDMIVTDNNGAVKFPQLCKF CDVRFST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMC S C S SDECNDNIIF SEEYNT SNPDIT CPPPM S VEFLADIW VK S YSL Y SRER YICN S GFKRK AGT S SLTEC VLNK ATN VAF1WTTP SLKCIR (SEQ ID NO: 132).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC

AACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT

CAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCC

ATCTGCGAGAAGCCCC AAGAAGT GTGCGT GGCCGTGT GGCGGAAAAAT GAC

GAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG

ACTTC ATTCTGGAGGACGCTGCCTCCCCC AAAT GC AT CAT GAAGGAGAAGAA

GAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAAC

GACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTG

GCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGT

GC AG A AG AGC GT G A AT A AT GAC AT GAT C GT GAC C GAT A AC A AT GGC GC C GT G

AAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA

CCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCT

C AGGAGGTGT GCGT GGCTGTCTGGCGGAAGAATGACGAGAAT ATC ACCCTGG

AAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGA

CGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGAC

CTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTA

GCGAGGAATACAATACCAGCAACCCCGACATCACGTGTCCTCCTCCTATGTC

CGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAG

CGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGAC

GGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGT

CTCAAATGTATTAGA (SEQ ID NO: 133).

Exemplary Multi-Chain Chimeric Polypeptides- Type C

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-7 or a receptor of IL-21. In some examples of these multi-chain chimeric polypeptides, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi - chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target-binding domain and the second target-binding domain is a soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or a soluble IL-7 (e.g., a soluble human IL-7 polypeptide). In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain are each independently a soluble IL-21 or a soluble IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain both bind specifically to a receptor of IL-21 or a receptor of IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain binds specifically to a receptor for IL-21, and the second target-binding domain binds specifically to a receptor for IL-7. In some embodiments of these multi- chain chimeric polypeptides, the first target-binding domain binds specifically to a receptor for IL-7, and the second target-binding domain binds specifically to a receptor for IL-21.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain includes a soluble IL-21 (e.g., a soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKS ANTGNNERIINV SIKKLKRKPP STNAGRRQKHRLTCP SCD S YEKKPPKEFLER FK SLLQKMIHQHL S SRTHGSED S (SEQ ID NO: 124). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTG ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCA GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGG C C C A AC T A A AGT C AGC A A AT AC AGG A A AC A AT G A A AGG AT A ATC A AT GT AT C AATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACA GAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCC AAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGC ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (SEQ ID NO: 134).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 125).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-7 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A ALGE AQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 135). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GATTGT GAT ATT GA AGGT A A AG AT GGC A A AC A AT AT GAGAGT GTTC T A AT GG T C AGC ATCGAT C A ATT ATT GGAC AGC AT GAAAGAA ATT GGT AGC AATTGCCT GAAT AAT GAATTT AACTTTTTT AAAAGAC AT ATCTGT GAT GCT AAT AAGGAA GGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAA T AGC ACTGGT GATTTT GATCTCC ACTT ATT AAAAGTTTC AGAAGGC AC AAC A A TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGG T GA AGCC C A AC C A AC A A AGAGTTT GGA AGA A A AT A A AT C TTT A A AGG A AC A GA A A A A AC T GAAT GAC TT GT GTTTCC T A A AG AG ACT ATT AC A AG AG AT A A A A ACTTGTT GGAAT AAAATTTTGATGGGC ACT AAAGAAC AC (SEQ ID NO: 136).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKS ANTGNNERIINV SIKKLKRKPP STNAGRRQKHRLTCP SCD S YEKKPPKEFLER FK SLLQKMIHQHL S SRTHGSED S S GTTNT V A A YNLTWK S TNFKTILEWEPKP VN Q VYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVEST GSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLS LRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNEFLID VDKGEN Y CF S VQ A VIP SRT VNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHP SCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECE ELEEKNIKEFLQ SF VHIV QMFINTS (SEQ ID NO: 126).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTG

ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCA GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGG

C C C A AC T A A AGT C AGC A A AT AC AGG A A AC A AT G A A AGG AT A ATC A AT GT AT C

AATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACA

GAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCC

AAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGC

ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTCAGGCACTACAAATAC

TGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGG

AGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAA

GTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGAC

CTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCT

TCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCT

GTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAG

CC AAC AATTC AGAGTTTTGAAC AGGT GGGAAC AAAAGT GAAT GTGACCGT AG

AAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGA

TGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTC

AGGAAAGAAAAC AGCC A AAAC AAAC ACT AAT GAGTTTTT GATT GAT GTGGAT

AAAGGAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAG

TTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAG

GGGA ATTC AGAGA A A AC T GGGT GA AC GT CAT C AGC GATTT A A AGA AGATCG

AAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGAC

GTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCA

AGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAAT

TTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTC

CGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCT

GCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO:

127).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: MGVKVLFALICIAVAEAQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEW SAF SCF QKAQLKS ANTGNNERIINV SIKKLKRKPPSTNAGRRQKHRLT CP S CD S YEKKPPKEFLERFK SLLQKMIHQHL S SRTHGSED S SGTTNT V A A YNLT W KSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQ T YL ARVF S YP AGNVE S T GS AGEPL YEN SPEF TP YLETNLGQPTIQ SFEQ V GTK VN VT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNEFLID V DKGEN Y CF S VQ A VIP SRT VNRK S TD SP VECMGQEKGEFRENW VN VI SDLKKIED LIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAN NSL S SN GN VTES GCKECEELEEKNIKEFLQ SF VHI V QMFINT S (SEQ ID NO: 128).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCCCA

AGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATC

AGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAA

GATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCA

ACT A A AGT C AGC A A AT AC AGGA A AC A AT GA A AGGAT A AT C A AT GT ATC A ATT

A A A A AGC T GA AGAGGA A ACC ACC TT C C AC A A AT GC AGGGAGA AG AC AGA A A

CACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAG

AATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTG

TCCTCTAGAACACACGGAAGTGAAGATTCCTCAGGCACTACAAATACTGTGG

C AGC AT AT AATTT AACTT GGAAAT C AACT AATTT C AAGAC AATTTTGGAGTGG

GAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAG

GAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCAC

CGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCC

TACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATG

AGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAAC

AATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGAT GAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTT TTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGA AAGAAAAC AGCC AAAAC AA AC ACT AAT GAGTTTTT GATTGAT GT GGAT AAAG GAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAAC CGGAAGAGT AC AGAC AGCCCGGT AGAGT GT AT GGGCC AGGAGAAAGGGGAA TTCAGAGAAAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATT TAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCAC CCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTAT CTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATC ATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCT GC A AGGAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AAT CCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 129)

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A ALGE AQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEH ADIWVKS Y SLY SRERYICNSGFKRK AGT S SLTEC VLNK ATNVAHWTTP SLKCIR

(SEQ ID NO: 137)

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGT GAT ATT GA AGGT A A AG AT GGC A A AC A AT AT GAGAGT GTTC T AAT GG T C AGC ATCGAT C A ATT ATT GGAC AGC AT GAAAGAA ATT GGT AGC AATTGCCT GAAT AAT GAATTT AACTTTTTT AAAAGAC AT ATCTGT GAT GCT AAT AAGGAA GGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAA T AGC ACTGGT GATTTT GATCTCC ACTT ATT AAAAGTTTC AGAAGGC AC AAC A A TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGG T GA AGCC C A AC C A AC A A AGAGTTT GGA AGA A A AT A A AT C TTT A A AGG A AC A GA A A A A AC T GA AT GAC TT GT GTTTCC T A A AG AG ACT ATT AC A AG AG AT A A A A ACTTGTT GGAAT AAAATTTTGAT GGGC ACT AAAGAAC AC AT C ACGT GCCCTC CCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTA CTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACG TCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGA CAACCCCCAGTCTCAAATGCATTAGA (SEQ ID NO: 138).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MGVKVLFALICIAVAEADCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN EFNFFKREQCD ANKEGMFLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLN C TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEHITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRKAGTS SLTECVLNK ATNVAHWTTPSLKCIR (SEQ ID NO: 139)

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCCGA T T GT GAT ATT G A AGGT A A AG AT GGC A A AC A AT AT G AG AGT GT TC T A AT GGT C AGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGA ATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGT ATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAG CACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATAC TGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGA AGC CC A AC C A AC A A AGAGTTT GGA AGA A A AT A A AT C TTT A A AGG A AC AGA A AAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACT TGTTGGAATAAAATTTTGATGGGCACTAAAGAACACATCACGTGCCCTCCCC CCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTC CAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCC AGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAA CCCCCAGTCTCAAATGCATTAGA (SEQ ID NO: 140). Exemplary Multi-Chain Chimeric Polypeptides- Type D

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-7 or a receptor of IL-21. In some examples of these multi-chain chimeric polypeptides, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target-binding domain and the second target-binding domain is a soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or a soluble IL-7 (e.g., a soluble human IL-7 polypeptide). In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain are each independently a soluble IL-21 or a soluble IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain both bind specifically to a receptor of IL-21 or a receptor of IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain binds specifically to a receptor for IL-21, and the second target-binding domain binds specifically to a receptor for IL-7. In some embodiments of these multi chain chimeric polypeptides, the first target-binding domain binds specifically to a receptor for IL-7, and the second target-binding domain binds specifically to a receptor for IL-2l.

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKS ANTGNNERIINV SIKKLKRKPP STNAGRRQKHRLTCP SCD S YEKKPPKEFLER FK SLLQKMIHQHL S SRTHGSED S (SEQ ID NO: 124).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTG ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCC AGCTCC A GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGG C C C A AC T A A AGT C AGC A A AT AC AGG A A AC A AT G A A AGG AT A ATC A AT GT AT C AATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACA GAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCC AAAGAATTCCTAGAAAGATTC AAATC ACTTCTCCAAAAGATGATTCATCAGC ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (SEQ ID NO: 134).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 125). In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-7 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP AALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 135). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGT GAT ATT GA AGGT A A AG AT GGC A A AC A AT AT GAGAGT GTTC T A AT GG T C AGC ATCGAT C A ATT ATT GGAC AGC AT GAAAGAA ATT GGT AGC AATTGCCT GAAT AAT GAATTT AACTTTTTT AAAAGAC AT ATCTGT GAT GCT AAT AAGGAA GGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAA T AGC ACTGGT GATTTT GATCTCC ACTT ATT AAAAGTTTC AGAAGGC AC AAC A A TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGG T GA AGCC C A AC C A AC A A AGAGTTT GGA AGA A A AT A A AT C TTT A A AGG A AC A GA A A A A AC T GAAT GAC TT GT GTTTCC T A A AG AG ACT ATT AC A AG AG AT A A A A ACTTGTT GGAAT AAAATTTTGATGGGC ACT AAAGAAC AC (SEQ ID NO: 136).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A ALGE AQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAY NLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVK DVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVG TK VN VT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNE FLID VDKGENY CF S VQ AVIPSRTVNRKSTD SPVECMGQEKGEFRENWVNVISDL KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVEN LIIL ANN SL S SN GN VTE S GCKECEELEEKNIKEFLQ SF VHI V QMFINT S (SEQ ID NO: 141).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG

GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC

TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA

GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG

AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA

CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT

GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA

GC AGAAGAAGCTGAACGACCTGT GCTTCCTGAAGAGGCTGCTGC AGGAGAT C

AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACA

ACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAA

CCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGAT

CAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACC

GAGT GCGATCTC ACCGAT GAGATCGTGAA AGATGT GAAAC AGACCT ACCTCG

CCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGG

CGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATT

T AGGAC AGCCC ACC ATCC AAAGCTTT GAGC A AGTT GGC AC AAAGGT GAAT GT

GACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGC

CTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTC CTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATC GACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCT CCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCA AGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAA GAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACA GAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACT GGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACC GTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGT GAC AGAGTCCGGCTGC A AGGAGT GCGAAGAGCTGGAGGAGAAGAAC AT C A A GGAGTTTCTGCAATCCTTTGTGCAC ATTGTCCAGATGTTCATCAATACCTCC

(SEQ ID NO: 142).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKW VTFISLLFLF S SAY SDCDIEGKDGKQ YES VLMV SIDQLLD SMKEIGSNCLNN EFNFFKRHICD ANKEGMFLFR A ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLN C TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEH S GTTNT V A A YNLT WK S TNFK TILEWEPKP VN Q V YT V QI S TK S GD WK S KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP YLETNLGQPTIQ SFEQ V GTKVNVTVEDERTLVRRNNTFL SLRD VF GKDLI YTL YY WKSS S SGKKTAKTNTNEFLIDVDKGENYCF S VQ A VIP SRT VNRK S TD SP VECMG QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL Q VI SLE S GD ASIHD T VENLIIL ANN SL S SN GN VTE S GCKECEELEEKNIKEFLQ SF V HIVQMFINTS (SEQ ID NO: 143).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGAT

GGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGC

CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG

AGGGC AT GTTCCTGTT C AGGGCCGCC AGGAAACTGCGGC AGTTCCTGAAGAT

GAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACC

ACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC

TGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG

AGC AGAAGAAGCTGAACGACCTGT GCTTCCTGAAGAGGCTGCTGC AGGAGAT

CAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCAC

AACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAA

ACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGA

TCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAC

CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTC

GCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTG

GCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAA

TTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAAT

GTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCA

GCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAG

TCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAA

TCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCC

CTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGC

CAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTA

AAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACA

CAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTA

CTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACA

CCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAAC

GT G AC AG AGT C C GGC T GC A AGG AGT GC G A AG AGC T GG AGG AG A AG A AC ATC AAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTC C (SEQ ID NO: 144).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKS ANTGNNERIINV SIKKLKRKPP STNAGRRQKHRLTCP SCD S YEKKPPKEFLER FK SLLQKMIHQHL S SRTHGSED SIT CPPPM S VEFLADIW VK S YSL Y SRERYICN S GF KRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 145).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG

ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC

CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG

GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG

AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG

CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC

CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA

GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTC

CCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAG

CCGGGAGAGGT AT ATCTGT AAC AGCGGCTT C AAGAGGAAGGCCGGC ACC AG

CAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACA

ACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 146).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEW SAF SCF QKAQLKS ANTGNNERIINV SIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIW VKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAF1WTTPSLKCIR (SEQ ID NO: 147).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CC AGGGCC AGGACAGGCAC ATGATCCGGATGAGGCAGCTC ATCGACATCGTC GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGT GAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAG GCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCC CCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATC AGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCT CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 148).

Exemplary Multi-Chain Chimeric Polypeptides- Type E

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor for IL-18 (e.g., a soluble human IL-18), a receptor for IL-12 (e.g., a soluble human IL-12), or CD16 (e.g., an anti-CDl6 scFv). In some embodiments of these multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to CD16 or a receptor for IL-12.

In some examples of these multi-chain chimeric polypeptides, the first target- binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of the first target-binding domain, the second target-binding domain and the additional antigen-binding domain is an agonistic antigen-binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain, the second target-binding domain, and the additional antigen-binding domain are each agonistic antigen-binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target-binding domain and the second target-binding domain is a soluble IL-15 or a soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain are each independently a soluble IL-15 or a soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain both bind specifically to a receptor of IL-18 or a receptor of IL-12. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain binds specifically to a receptor for IL-12, and the second target-binding domain binds specifically to a receptor for IL-18. In some embodiments of these multi- chain chimeric polypeptides, the first target-binding domain binds specifically to a receptor for IL-18, and the second target-binding domain bind specifically to a receptor for IL-12. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain binds specifically to CD 16, and the second target-binding domain binds specifically to a receptor for IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain binds specifically to a receptor for IL-18, and the second target-binding domain bind specifically to CD 16.

In some embodiments of these multi-chain chimeric polypeptides, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain includes a soluble IL-18 (e.g., a soluble human IL-18).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-18 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQ PRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKM QFES S S YEGYFL ACEKERDLFKLILKKEDELGDRSIMFT V QNED (SEQ ID NO: 41).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-18 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACC AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGAC ATGACCGAC TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACC CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC GTGCCCGGTCACGATAACAAGATGC AGTTCGAATCCTCCTCCTACGAGGGCT ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT

(SEQ ID NO: 42).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain includes a soluble IL-12 (e.g., a soluble human IL-12). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-15 includes a sequence of soluble human IL-l2p (p40) and a sequence of soluble human IL- 12a (p35). In some embodiments of these multi-chain chimeric polypeptides, the soluble IL-15 (e.g., soluble human IL-15) further includes a linker sequence (e.g., any of the exemplary linker sequences described herein) between the sequence of soluble IL-l2p

(p40) and the sequence of soluble human IL-l2a (p35). In some examples of these multi- chain chimeric polypeptides, the linker sequence comprises GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-l2p (p40) comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKEF GD AGQ YTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQGVTCGAATLS AERVRGDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKN LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT SATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 33).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-l2p (p40) is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA AGGGAGGCGAGGT GCTC AGCC ATTCCTT ATT ATT ATT AC AC AAGAAGGAAGA CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGC T GGTCCGAGT GGGCC AGCGT GCCTT GTTCC (SEQ ID NO: 34).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-l2a (p35) includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKD KTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKM Y QVEFKTMNAKLLMDPKRQIFLDQNML AVIDELMQ ALNFN SETVPQKS SLEEPD FYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 35).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-l2a (p35) is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCA CAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAG ACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCA CCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAA GAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTT GTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCT ACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCT GCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTG ATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGA AGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCAT TTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCT ATTTAAACGCCAGC (SEQ ID NO: 36). In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain includes an scFv that specifically binds to CD 16 (e.g., an anti- CD 16 scFv).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a light chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRS YYASWYQQKPGQAPVLVIYGKNNRPS GIPDRF S GS S S GNT ASLTIT GAQ AEDE AD Y Y CN SRD S S GNH VVF GGGTKLT V GH

(SEQ ID NO: 149).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a light chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GC AG A AGC C C GGC C AGGC TC C T GT GC T GGT GAT C T AC GGC A AG A AC A AC AGG CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCAT (SEQ ID NO: 150).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a heavy chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINW NGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDY WGQGTLVTVSR (SEQ ID NO: 151).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a heavy chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

G AGGT GC AGC T GGT GG AGT C C GG AGG AGG AGT GGT G AGGC C T GG AGGC T C C

CTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTC

CTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAAC

TGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCA

TCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAG

GGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTC

GACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 152).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQ

PRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKM

QFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYN

LTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKD

VKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGT

K VN VT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNEF

LID VDKGEN Y CF S VQ A VIP SRT VNRK S TD SP VECMGQEKGEFRENW VN VISDLK KIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLI IL ANN SL S SN GN VTE S GCKECEELEEKNIKEFLQ SF VHI V QMFINT S (SEQ ID NO: 116).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACC

AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGAC

TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA

GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA

AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACC

CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC

GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT

ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA

GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA

ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC

CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACC

ACCGAC ACCGAGT GCGATCTC ACCGAT GAGATCGT GAAAGAT GT GAAAC AGA

CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG

TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG

AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA

GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACAC

CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT

ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA

GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT

GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT

GC AT GGGCC AAGAA AAGGGCGAGTTCCGGGAGAACTGGGT GAACGT CAT C A

GCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCAC TTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT _{GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATC} CACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAA CGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAA GAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCA ATACCTCC (SEQ ID NO: 1 17).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD CRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKD TKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM F TV QNED S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QI STK S GD WK S KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP YLETNLGQPTIQ SFEQ V GTKVNVTVEDERTLVRRNNTFL SLRD VF GKDLI YTL YY WKSS S SGKKTAKTNTNEFLIDVDKGENYCF S VQ A VIP SRT VNRK S TD SP VECMG QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL Q VI SLE S GD ASIHD T VENLIIL ANN SL S SN GN VTE S GCKECEELEEKNIKEFLQ SF V HIVQMFINTS (SEQ ID NO: 1 18).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACC

AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGAC

TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA

AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACC

CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC

GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT

ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA

GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA

ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC

CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACC

ACCGAC ACCGAGT GCGATCTC ACCGAT GAGATCGT GAAAGAT GT GAAAC AGA

CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG

TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG

AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA

GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACAC

CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT

ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA

GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT

GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT

GC AT GGGCC AAGAA AAGGGCGAGTTCCGGGAGAACTGGGT GAACGT CAT C A

GCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCAC

TTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT

GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATC

CACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAA

CGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAA

GAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCA

ATACCTCC (SEQ ID NO: 119).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKEF GD AGQ YTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQGVTCGAATLS AERVRGDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKN LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT SATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPV ATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTV EACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEF KTMNAKLLMDPKRQIFLDQNMLAVIDELMQ ALNFN SETVPQKS SLEEPDFYKTK IKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICN S GFKRK AGT S SLTEC VLNKATN V AHWTTP SLKCIRSELT QDP A V S V ALGQ T VRIT CQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITG AQ AEDE AD Y Y CN SRD S S GNH VVF GGGTKLT V GHGGGGS GGGGS GGGGSE V QL VESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGG STGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQ GTLVTVSR (SEQ ID NO: 153).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG

ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG

CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC

CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA

AGGGAGGCGAGGT GCTC AGCC ATTCCTT ATT ATT ATT AC AC AAGAAGGAAGA

CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG

ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG

GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA

AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA

GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC

GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA

CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA

GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA

CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG

AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG

GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGC

TGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAG

GTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCC

CGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGC

AACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCG

AGGAGATCGACC AT GAAGAT AT C ACC AAGGAC AAGAC ATCC ACCGT GGAGG

CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAA

ACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTAT

GATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGG

AGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTT

TTT AGACC AGAAC AT GCTGGCTGT GATT GAT GAGCTGATGC AAGCTTT AAACT

TCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTAC

AAGAC AAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCG

TGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCT

CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA

GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA

GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC

AACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGT

CCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAG

GTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTG

GTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCG

GATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGA

GGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTG

GTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCG

GAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCG GAGGAGGAGTGGT GAGGCCTGGAGGCTCCCTGAGGCTGAGCTGT GCTGCCTC

CGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGA

AAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCT

ACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGA

ACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTA

CTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACC

CTGGTGACCGTGTCCAGG (SEQ ID NO: 154).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKW VTFISLLFLF S SAY SIWELKKD VYVVELDW YPD APGEMVVLT CDTPEEDGI TWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQG VTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYE NYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQV QGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGG GSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYP CT SEEIDHEDITKDKT S T VE ACLPLELTKNE S CLN SRET SFITN GS CL ASRKT SFMM ALCLS SIYEDLKMY Q VEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQ ALNFN S ETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHA DIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSE LTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGI PDRFSGS S SGNTASLTITGAQ AEDEAD YY CN SRDS SGNHVVF GGGTKLT VGHGG GGS GGGGS GGGGSE V QL VES GGGVVRPGGSLRL S C A AS GF TFDD Y GMS W VRQ A PGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV YY C ARGRSLLFD YW GQGTL VT V SR (SEQ ID NO: 155).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG

ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG

CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC

CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA

AGGGAGGCGAGGT GCTC AGCC ATTCCTT ATT ATT ATT AC AC AAGAAGGAAGA

CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG

ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG

GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA

AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA

GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG

ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC

GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA

CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA

GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA

CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG

AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG

GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGC

TGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAG

GTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCC

CGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGC

AACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCG

AGGAGATCGACC AT GAAGAT AT C ACC AAGGAC AAGAC ATCC ACCGT GGAGG

CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAA

ACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTAT

GATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGG

AGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTT

TTT AGACC AGAAC AT GCTGGCTGT GATT GAT GAGCTGATGC AAGCTTT AAACT TCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTAC

AAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCG

TGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCT

CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA

GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA

GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC

AACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGT

CCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAG

GTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTG

GTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCG

GATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGA

GGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTG

GTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCG

GAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCG

GAGGAGGAGTGGT GAGGCCTGGAGGCTCCCTGAGGCTGAGCTGT GCTGCCTC

CGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGA

AAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCT

ACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGA

ACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTA

CTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACC

CTGGTGACCGTGTCCAGG (SEQ ID NO: 156).

Exemplary Multi-Chain Chimeric Polypeptides- Type F

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor for IL-7 (e.g., a soluble human IL-7), CD 16 (e.g., an anti-CDl6 scFv), or a receptor for IL-21 (e.g., a soluble human IL-21). In some embodiments of these multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to CD 16 or a receptor for IL-21.

In some examples of these multi-chain chimeric polypeptides, the first target- binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of the first target-binding domain, the second target-binding domain and the additional antigen-binding domain is an agonistic antigen-binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain, the second target-binding domain, and the additional antigen-binding domain are each agonistic antigen-binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain binds specifically to a receptor IL-7 and the second target-binding domain binds specifically to CD 16 or a receptor for IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain includes a soluble IL-7 protein. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-7 protein is a soluble human IL-7. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second antigen-binding domain includes a target binding domain that binds specifically to CD 16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain includes an scFv that binds specifically to CD 16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain binds specifically to a receptor for IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain includes a soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-21 is a soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes an additional target-binding domain that binds specifically to a receptor for IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the additional target-binding domain includes a soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-21 is a soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes an additional target-binding domain that binds specifically to CD 16.

In some embodiments of these multi-chain chimeric polypeptides, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain includes a soluble IL-7 (e.g., a soluble human IL-7).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP AALGE AQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO : 135). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGT GAT ATT GA AGGT A A AG AT GGC A A AC A AT AT GAGAGT GTTC T A AT GG T C AGC ATCGAT C A ATT ATT GGAC AGC AT GAAAGAA ATT GGT AGC AATTGCCT GAAT AAT GAATTT AACTTTTTT AAAAGAC AT ATCTGT GAT GCT AAT AAGGAA GGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAA T AGC ACTGGT GATTTT GATCTCC ACTT ATT AAAAGTTTC AGAAGGC AC AAC A A TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGG T GA AGCC C A AC C A AC A A AGAGTTT GGA AGA A A AT A A AT C TTT A A AGG A AC A GA A A A A AC T GAAT GAC TT GT GTTTCC T A A AG AG ACT ATT AC A AG AG AT A A A A ACTTGTT GGAAT AAAATTTTGATGGGC ACT AAAGAAC AC (SEQ ID NO: 136). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GC AGAAGAAGCTGAACGACCTGT GCTTCCTGAAGAGGCTGCTGC AGGAGAT C AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO: 197). In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-21 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKS ANTGNNERIINV SIKKLKRKPP STNAGRRQKHRLTCP SCD S YEKKPPKEFLER FK SLLQKMIHQHL S SRTHGSED S (SEQ ID NO: 124).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTG ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCC AGCTCC A GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGG C C C A AC T A A AGT C AGC A A AT AC AGG A A AC A AT G A A AGG AT A ATC A AT GT AT C AATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACA GAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCC AAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGC ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (SEQ ID NO: 134).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 125) In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain includes an scFv that specifically binds to CD 16 (e.g., an anti- CD 16 scFv).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a light chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRF S GS S S GNT ASLTIT GAQ AEDE AD Y Y CN SRD S S GNH VVF GGGTKLT V GH

(SEQ ID NO: 149).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a light chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GC AG A AGC C C GGC C AGGC TC C T GT GC T GGT GAT C T AC GGC A AG A AC A AC AGG CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCAT (SEQ ID NO: 150). In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a heavy chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINW NGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDY WGQGTLVTVSR (SEQ ID NO: 151).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a heavy chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

G AGGT GC AGC T GGT GG AGT C C GG AGG AGG AGT GGT G AGGC C T GG AGGC T C C

CTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTC

CTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAAC

TGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCA

TCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAG

GGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTC

GACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 152).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A ALGE AQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAY NLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVK DVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVG TK VN VT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNE FLID VDKGENY CF S VQ AVIPSRTVNRKSTD SPVECMGQEKGEFRENWVNVISDL KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVEN LIIL ANN SL S SN GN VTE S GCKECEELEEKNIKEFLQ SF VHI V QMFINT S (SEQ ID NO: 141).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG

GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC

TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA

GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG

AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA

CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT

GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA

GC AGAAGAAGCTGAACGACCTGT GCTTCCTGAAGAGGCTGCTGC AGGAGAT C

AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACA

ACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAA

CCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGAT

CAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACC

GAGT GCGATCTC ACCGAT GAGATCGTGAA AGATGT GAAAC AGACCT ACCTCG

CCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGG

CGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATT

T AGGAC AGCCC ACC ATCC AAAGCTTT GAGC A AGTT GGC AC AAAGGT GAAT GT

GACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGC

CTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTC

CTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATC GACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCT

CCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCA

AGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAA

GAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACA

GAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACT

GGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACC

GTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGT

GAC AGAGTCCGGCTGC A AGGAGT GCGAAGAGCTGGAGGAGAAGAAC AT C A A

GGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

(SEQ ID NO: 142).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKW VTFISLLFLF S SAY SDCDIEGKDGKQ YES VLMV SIDQLLD SMKEIGSNCLNN EFNFFKRHICD ANKEGMFLFR A ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLN C TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEH S GTTNT V A A YNLT WK S TNFK TILEWEPKP VN Q V YT V QI S TK S GD WK S KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP YLETNLGQPTIQ SFEQ V GTKVNVTVEDERTLVRRNNTFL SLRD VF GKDLI YTL YY WKSS S SGKKTAKTNTNEFLIDVDKGENYCF S VQ A VIP SRT VNRK S TD SP VECMG QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL Q VI SLE S GD ASIHD T VENLIIL ANN SL S SN GN VTE S GCKECEELEEKNIKEFLQ SF V HIVQMFINTS (SEQ ID NO: 143).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGAT

GGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGC

CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG

AGGGC AT GTTCCTGTT C AGGGCCGCC AGGAAACTGCGGC AGTTCCTGAAGAT

GAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACC

ACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC

TGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG

AGC AGAAGAAGCTGAACGACCTGT GCTTCCTGAAGAGGCTGCTGC AGGAGAT

CAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCAC

AACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAA

ACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGA

TCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAC

CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTC

GCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTG

GCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAA

TTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAAT

GTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCA

GCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAG

TCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAA

TCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCC

CTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGC

CAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTA

AAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACA

CAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTA

CTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACA

CCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAAC

GT G AC AG AGT C C GGC T GC A AGG AGT GC G A AG AGC T GG AGG AG A AG A AC ATC

AAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTC

C (SEQ ID NO: 144). In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRF S GS S S GNT ASLTIT GAQ AEDE AD Y Y CN SRD S S GNH VVF GGGTKLT V GHG GGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQ APGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTA VYY C ARGRSLLFD YW GQGTL VT V SRIT CPPPMS VEHADIWVKS Y SLY SRERYIC N S GFKRK AGT S SLTEC VLNK ATN V AF1WTTP SLKCIRQ GQDRHMIRMRQLIDI VD QLKNYVNDLVPEFLP APEDVETNCEW S AF SCF QKAQLKS ANTGNNERIINV SIKK LKRKPP S TN AGRRQKHRLTCP S CD S YEKKPPKEFLERFK SLLQKMIHQHL S SRTH GSEDS (SEQ ID NO: 157).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA

GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA

GC AG A AGC C C GGC C AGGC TC C T GT GC T GGT GAT C T AC GGC A AG A AC A AC AGG

CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC

CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC

TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA

CCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAG

GATCCGAGGTGC AGCTGGT GGAGTCCGGAGGAGGAGT GGT GAGGCCTGGAG

GCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGC

ATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCA

TCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTT

CACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCC CTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGC

TGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATG

CCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGC

CTCT AC AGCCGGGAGAGGT AT ATCT GT AAC AGCGGCTT C AAGAGGAAGGCCG

GCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCA

CTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATG

ATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGA

ACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTG

CGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAAC

ACCGGC A AC AACGAGCGGAT CAT C AACGT GAGC AT C A AGAAGCTGAAGCGG

AAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGC

CCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGT

TCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCA

CGGCTCCGAGGACTCC (SEQ ID NO: 158).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQ KPGQ AP VL VI Y GKNNRP S GIPDRF S GS S SGNT ASLTIT GAQ AEDE AD Y Y CN SRD S S GNH VVF GGGTKLT V GHGGGGS GGGGS GGGGSE V QL VES GGGVVRPGGSLRL S C AASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNA KN SL YLQMN SLRAEDT AVYY C ARGRSLLFD YW GQGTL VT V SRITCPPPMS VEH ADIWVKS Y SLY SRERYICNSGFKRK AGT S SLTEC VLNK ATNVAHWTTP SLKCIRQ GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLP APEDVETNCEW S AF SCF QKAQL K S ANT GNNERIIN V SIKKLKRKPP S TN AGRRQKHRLT CP S CD S YEKKPPKEFLERF K SLLQKMIHQHL S SRTHGSED S (SEQ ID NO: 159).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTG

AGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACC

AGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAG

GCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCT

CCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAA

CTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTG

ACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGA

GG AT C C G AGGT GC AGC T GGT GG AGT C C GG AGG AGG AGT GGT G AGGC C T GG A

GGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGG

CAT GT C C T GGGT G AGGC AGGC TC C T GG A A AGGGC C T GG AGT GGGT GT C C GGC

ATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGT

TCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTC

CCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTG

CTGTTCGACTACTGGGGAC AGGGC ACCCTGGTGACCGTGTCCAGGATTAC AT

GCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAG

CCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCC

GGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTC

ACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACAT

GATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTG

AACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACT

GCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAA

CACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCG

GAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTG

CCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGG

TTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCC

ACGGCTCCGAGGACTCC (SEQ ID NO: 160). Exemplary Multi-Chain Chimeric Polypeptides- Type G

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to TGF-b (e.g., a human TGFpRII receptor), CD16 (e.g., an anti-CDl6 scFv), or a receptor for IL-21 (e.g., a soluble human IL-21). In some embodiments of these multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to CD 16 or a receptor for IL-21.

In some examples of these multi-chain chimeric polypeptides, the first target- binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of the first target-binding domain, the second target-binding domain and the additional antigen-binding domain is an agonistic antigen-binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain, the second target-binding domain, and the additional antigen-binding domain are each agonistic antigen-binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to TGF-b, CD 16, or a receptor for IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain binds specifically to a TGF-b and the second target-binding domain binds specifically to CD 16 or a receptor of IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain is a soluble TGF-b receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, soluble TGF-b receptor is a soluble TORbIIP receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain binds specifically to CD 16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second antigen-binding domain includes an antigen-binding domain that binds specifically to CD 16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second antigen-binding domain includes an scFv that binds specifically to CD 16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain binds specifically to a receptor for IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain includes a soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain includes a soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes an additional target-binding domain that binds specifically to a receptor for IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the additional target-binding domain includes a soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-21 is a soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes an additional target- binding domain that binds specifically to CD 16.

In some embodiments of these multi-chain chimeric polypeptides, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain includes a TGF pRI I receptor (e.g., a soluble human TGF pRII receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-PRII includes a first sequence of soluble human TGF-b RII and a second sequence of soluble human TGF-b RII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII includes a linker disposed between the first sequence of soluble human TGF-b RII and the second sequence of soluble human TGF-b RII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56). In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA

ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC

AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA

TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG

AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA

CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG

AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG

ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO:

57)

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGT GAGAAGCCTC AGGAGGT GTGCGT GGCTGTCTGGCGGAAGAAT GACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO:

57).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-b receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIF SEE YNT SNPD (SEQ ID NO: 60).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 198). In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-21 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKS ANTGNNERIINV SIKKLKRKPP STNAGRRQKHRLTCP SCD S YEKKPPKEFLER FK SLLQKMIHQHL S SRTHGSED S (SEQ ID NO: 124).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTG ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCC AGCTCC A GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGG C C C A AC T A A AGT C AGC A A AT AC AGG A A AC A AT G A A AGG AT A ATC A AT GT AT C AATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACA GAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCC AAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGC ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (SEQ ID NO: 134).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 125). In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a light chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRF S GS S S GNT ASLTIT GAQ AEDE AD Y Y CN SRD S S GNH VVF GGGTKLT V GH

(SEQ ID NO: 149).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a light chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GC AG A AGC C C GGC C AGGC TC C T GT GC T GGT GAT C T AC GGC A AG A AC A AC AGG CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCAT (SEQ ID NO: 150).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a heavy chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINW NGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDY WGQGTLVTVSR (SEQ ID NO: 151).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a heavy chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

G AGGT GC AGC T GGT GG AGT C C GG AGG AGG AGT GGT G AGGC C T GG AGGC T C C

CTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTC

CTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAAC

TGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCA

TCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAG

GGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTC

GACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 152).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE

KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET

FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM

IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK

NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND

NIIF SEEYNT SNPD S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QI S TK S

GDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYEN SPEF TP YLETNLGQPTIQ SFEQ V GTK VN VT VEDERTL VRRNNTFL SLRD VF GKDLI YTLYYWKSS S SGKKTAKTNTNEFLIDVDKGENYCF S VQ AVIPSRTVNRKSTDSP V ECMGQEKGEFRENW VN VI SDLKKIEDLIQ SMHID ATL YTESD VHP S CK VT AMKC FLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS (SEQ ID NO: 161).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA

ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC

AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA

TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG

AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA

CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG

AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG

ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG

CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG

CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA

AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC

CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC

AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA

AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC

GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC

TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG

CGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGC

TGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGG

GAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCG

GCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCAC

CGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGC TACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACG

AGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCAC

C ATCC AAAGCTTT GAGC AAGTT GGC AC AAAGGTGAAT GT GAC AGT GGAGGAC

GAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGT

TCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC

AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAA

GGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGA

ATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCG

AGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGA

TTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGC

ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGC AAGTT

ATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAA

TCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGC

T GC A AGGAGT GC GA AGAGCTGGAGGAGA AGA AC AT C A AGGAGTTTC T GCA A

TCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 162).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S SAY SIPPHV QKS VNNDMIVTDNNGAVKFPQLCKF CDVRFST

CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED

AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG

GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI

TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK

PGETFFMC S C S SDECNDNIIF SEEYNT SNPD SGTTNT V A A YNLT WK S TNFKTILEW

EPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYP

AGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLV

RRNNTFLSLRD VFGKDLIYTL YYWKS S S SGKKT AKTNTNEFLID VDKGEN Y CF S V

Q A VIP SRT VNRK S TD SP VECMGQEKGEFRENW VN VI SDLKKIEDLIQ SMHID ATL YTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVT ESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 163).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC

AACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT

CAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCC

ATCTGCGAGAAGCCCC AAGAAGT GTGCGT GGCCGTGT GGCGGAAAAAT GAC

GAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG

ACTTC ATTCTGGAGGACGCTGCCTCCCCC AAAT GC AT CAT GAAGGAGAAGAA

GAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAAC

GACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTG

GCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGT

GC AG A AG AGC GT G A AT A AT GAC AT GAT C GT GAC C GAT A AC A AT GGC GC C GT G

AAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA

CCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC

AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA

AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC

GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC

TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG

CGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGC

TGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGG

GAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCG

GCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCAC

CGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGC

TACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACG

AGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCAC C ATCC AAAGCTTT GAGC AAGTT GGC AC AAAGGTGAAT GT GAC AGT GGAGGAC

GAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGT

TCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC

AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAA

GGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGA

ATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCG

AGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGA

TTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGC

ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGC AAGTT

ATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAA

TCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGC

T GC A AGGAGT GC GA AGAGCTGGAGGAGA AGA AC AT C A AGGAGTTTC T GCA A

TCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 164).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRF S GS S S GNT ASLTIT GAQ AEDE AD Y Y CN SRD S S GNH VVF GGGTKLT V GHG GGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQ APGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTA VYY C ARGRSLLFD YW GQGTL VT V SRIT CPPPMS VEHADIWVKS Y SLY SRERYIC N S GFKRK AGT S SLTEC VLNK ATN V AF1WTTP SLKCIRQ GQDRHMIRMRQLIDI VD QLKNYVNDLVPEFLP APEDVETNCEW S AF SCF QKAQLKS ANTGNNERIINV SIKK LKRKPP S TN AGRRQKHRLTCP S CD S YEKKPPKEFLERFK SLLQKMIHQHL S SRTH GSEDS (SEQ ID NO: 157).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA

GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA

GC AG A AGC C C GGC C AGGC TC C T GT GC T GGT GAT C T AC GGC A AG A AC A AC AGG

CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC

CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC

TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA

CCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAG

GATCCGAGGTGC AGCTGGT GGAGTCCGGAGGAGGAGT GGT GAGGCCTGGAG

GCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGC

ATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCA

TCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTT

CACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCC

CTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGC

TGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATG

CCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGC

CTCT AC AGCCGGGAGAGGT AT ATCT GT AAC AGCGGCTT C AAGAGGAAGGCCG

GCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCA

CTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATG

ATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGA

ACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTG

CGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAAC

ACCGGC A AC AACGAGCGGAT CAT C AACGT GAGC AT C A AGAAGCTGAAGCGG

AAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGC

CCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGT

TCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCA

CGGCTCCGAGGACTCC (SEQ ID NO: 158).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQ KPGQ AP VL VI Y GKNNRP S GIPDRF S GS S SGNT ASLTIT GAQ AEDE AD Y Y CN SRD S S GNH VVF GGGTKLT V GHGGGGS GGGGS GGGGSE V QL VES GGGVVRPGGSLRL S C AASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNA KN SL YLQMN SLRAEDT AVYY C ARGRSLLFD YW GQGTL VT V SRITCPPPMS VEH ADIWVKS Y SLY SRERYICNSGFKRK AGT S SLTEC VLNK ATNVAHWTTP SLKCIRQ GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLP APEDVETNCEW S AF SCF QKAQL K S ANT GNNERIIN V SIKKLKRKPP S TN AGRRQKHRLT CP S CD S YEKKPPKEFLERF K SLLQKMIHQHL S SRTHGSED S (SEQ ID NO: 159).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTG

AGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACC

AGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAG

GCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCT

CCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAA

CTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTG

ACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGA

GG AT C C G AGGT GC AGC T GGT GG AGT C C GG AGG AGG AGT GGT G AGGC C T GG A

GGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGG

CAT GT C C T GGGT G AGGC AGGC TC C T GG A A AGGGC C T GG AGT GGGT GT C C GGC

ATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGT

TCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTC

CCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTG

CTGTTCGACTACTGGGGAC AGGGC ACCCTGGTGACCGTGTCCAGGATTAC AT GCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAG CCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCC GGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTC ACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACAT GATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTG AACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACT GCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAA CACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCG GAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTG CCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGG TTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCC ACGGCTCCGAGGACTCC (SEQ ID NO: 160).

Exemplary Multi-Chain Chimeric Polypeptides- Type H

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-7. In some examples of these multi- chain chimeric polypeptides, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain and the second target-binding domain each independently bind specifically to a receptor for IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain and the second target-binding domain include a soluble IL-7 (e.g., a soluble human IL-7). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP AALGE AQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 135). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG

GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC

TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA

GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG

AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA

CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT

GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA

GC AGAAGAAGCTGAACGACCTGT GCTTCCTGAAGAGGCTGCTGC AGGAGAT C

AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO:

197).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP AALGE AQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAY NLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVK DVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVG TK VN VT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNE FLID VDKGENY CF S VQ AVIPSRTVNRKSTD SPVECMGQEKGEFRENWVNVISDL KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVEN LIIL ANN SL S SN GN VTE S GCKECEELEEKNIKEFLQ SF VHI V QMFINT S (SEQ ID NO: 141).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG

GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC

TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA

GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG

AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA

CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT

GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA

GC AGAAGAAGCTGAACGACCTGT GCTTCCTGAAGAGGCTGCTGC AGGAGAT C

AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACA

ACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAA

CCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGAT

CAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACC

GAGT GCGATCTC ACCGAT GAGATCGTGAA AGATGT GAAAC AGACCT ACCTCG

CCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGG

CGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATT

T AGGAC AGCCC ACC ATCC AAAGCTTT GAGC A AGTT GGC AC AAAGGT GAAT GT

GACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGC

CTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTC

CTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATC

GACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCT

CCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCA

AGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAA

GAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACA

GAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACT

GGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACC

GTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGT

GAC AGAGTCCGGCTGC A AGGAGT GCGAAGAGCTGGAGGAGAAGAAC AT C A A GGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 142).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKW VTFISLLFLF S SAY SDCDIEGKDGKQ YES VLMV SIDQLLD SMKEIGSNCLNN EFNFFKRHICD ANKEGMFLFR A ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLN C TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEH S GTTNT V A A YNLT WK S TNFK TILEWEPKP VN Q V YT V QI S TK S GD WK S KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP YLETNLGQPTIQ SFEQ V GTKVNVTVEDERTLVRRNNTFL SLRD VF GKDLI YTL YY WKSS S SGKKTAKTNTNEFLIDVDKGENYCF S VQ A VIP SRT VNRK S TD SP VECMG QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL Q VI SLE S GD ASIHD T VENLIIL ANN SL S SN GN VTE S GCKECEELEEKNIKEFLQ SF V HIVQMFINTS (SEQ ID NO: 143).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGAT

GGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGC

CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG

AGGGC AT GTTCCTGTT C AGGGCCGCC AGGAAACTGCGGC AGTTCCTGAAGAT

GAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACC

ACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC

TGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG AGC AGAAGAAGCTGAACGACCTGT GCTTCCTGAAGAGGCTGCTGC AGGAGAT

CAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCAC

AACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAA

ACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGA

TCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAC

CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTC

GCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTG

GCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAA

TTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAAT

GTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCA

GCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAG

TCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAA

TCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCC

CTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGC

CAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTA

AAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACA

CAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTA

CTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACA

CCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAAC

GT G AC AG AGT C C GGC T GC A AGG AGT GC G A AG AGC T GG AGG AG A AG A AC ATC

AAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTC

C (SEQ ID NO: 144).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A ALGE AQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEH ADIWVKS Y SLY SRERYICNSGFKRK AGT S SLTEC VLNKATNVAF1WTTP SLKCIR (SEQ ID NO: 137).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGT GAT ATT GA AGGT A A AG AT GGC A A AC A AT AT GAGAGT GTTC T A AT GG T C AGC ATCGAT C A ATT ATT GGAC AGC AT GAAAGAA ATT GGT AGC AATTGCCT GAAT AAT GAATTT AACTTTTTT AAAAGAC AT ATCTGT GAT GCT AAT AAGGAA GGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAA T AGC ACTGGT GATTTT GATCTCC ACTT ATT AAAAGTTTC AGAAGGC AC AAC A A TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGG T GA AGCC C A AC C A AC A A AGAGTTT GGA AGA A A AT A A AT C TTT A A AGG A AC A GA A A A A AC T GAAT GAC TT GT GTTTCC T A A AG AG ACT ATT AC A AG AG AT A A A A ACTTGTT GGAAT AAAATTTTGAT GGGC ACT AAAGAAC AC AT C ACGT GCCCTC CCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTA CTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACG TCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGA CAACCCCCAGTCTCAAATGCATTAGA (SEQ ID NO: 138).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKW VTFISLLFLF S SAY SDCDIEGKDGKQ YES VLMV SIDQLLD SMKEIGSNCLNN EFNFFKRHICD ANKEGMFLFR A ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLN C TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEHITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRKAGTS SLTECVLNK ATNVAHWTTPSLKCIR (SEQ ID NO: 139). In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGAT GGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGC CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG AGGGC AT GTTCCTGTT C AGGGCCGCC AGGAAACTGCGGC AGTTCCTGAAGAT GAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACC ACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC TGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG AGC AGAAGAAGCTGAACGACCTGT GCTTCCTGAAGAGGCTGCTGC AGGAGAT C AAGACCTGCTGGAAC AAGATCCTGATGGGC ACC AAGGAGC AT ATT AC AT GC CCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCC TCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGG CACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCAC TGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 140). Exemplary Multi-Chain Chimeric Polypeptides- Type I

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to TGF-b. In some examples of these multi-chain chimeric polypeptides, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain and the second target-binding domain each independently bind specifically to TGF-b. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain is a soluble TGF-b receptor (e.g., a soluble TGF-b RII receptor, e.g., a soluble human TGF-b RII). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII includes a first sequence of soluble human TGF^RII and a second sequence of soluble human TGF-b RII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-PRII includes a linker disposed between the first sequence of soluble human TGF-b RII and the second sequence of soluble human TGF-b RII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGS GGGGS GGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 57)

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGT GAGAAGCCTC AGGAGGT GTGCGT GGCTGTCTGGCGGAAGAAT GACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO:

57).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-b receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIF SEE YNT SNPD (SEQ ID NO: 60). In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-b receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGAC (SEQ ID NO: 198).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE

KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET

FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIF SEEYNT SNPD S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QI S TK S GDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYEN SPEF TP YLETNLGQPTIQ SFEQ V GTK VN VT VEDERTL VRRNNTFL SLRD VF GKDLI YTLYYWKS S S SGKKTAKTNTNEFLIDVDKGENYCF S VQ AVIPSRTVNRKSTDSP V ECMGQEKGEFRENW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKC FLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS (SEQ ID NO: 161).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA

ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC

AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA

TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG

AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA

CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG

AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG

ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG

CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG

CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA

AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC

CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC

AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA

AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC

GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC

TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG

CGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGC TGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGG

GAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCG

GCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCAC

CGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGC

TACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACG

AGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCAC

C ATCC AAAGCTTT GAGC AAGTT GGC AC AAAGGTGAAT GT GAC AGT GGAGGAC

GAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGT

TCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC

AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAA

GGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGA

ATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCG

AGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGA

TTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGC

ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGC AAGTT

ATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAA

TCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGC

T GC A AGGAGT GC GA AGAGCTGGAGGAGA AGA AC AT C A AGGAGTTTC T GCA A

TCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 162).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S SAY SIPPHV QKS VNNDMIVTDNNGAVKFPQLCKF CDVRFST

CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED

AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG

GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI

TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK

PGETFFMC S C S SDECNDNIIF SEEYNT SNPD SGTTNT V A A YNLT WK S TNFKTILEW

EPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYP AGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLV RRNNTFLSLRD VFGKDLIYTL YYWKS S S SGKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQEKGEFRENW VN VI SDLKKIEDLIQ SMHID ATL YTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVT ESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 163).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC

AACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT

CAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCC

ATCTGCGAGAAGCCCC AAGAAGT GTGCGT GGCCGTGT GGCGGAAAAAT GAC

GAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG

ACTTC ATTCTGGAGGACGCTGCCTCCCCC AAAT GC AT CAT GAAGGAGAAGAA

GAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAAC

GACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTG

GCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGT

GC AG A AG AGC GT G A AT A AT GAC AT GAT C GT GAC C GAT A AC A AT GGC GC C GT G

AAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA

CCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC

AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA

AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC

GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC

TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG

CGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGC

TGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGG

GAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCG

GCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCAC CGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGC

TACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACG

AGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCAC

C ATCC AAAGCTTT GAGC AAGTT GGC AC AAAGGTGAAT GT GAC AGT GGAGGAC

GAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGT

TCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC

AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAA

GGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGA

ATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCG

AGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGA

TTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGC

ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGC AAGTT

ATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAA

TCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGC

T GC A AGGAGT GC GA AGAGCTGGAGGAGA AGA AC AT C A AGGAGTTTC T GCA A

TCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 164).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE

KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET

FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM

IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK

NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND

NIIF SEEYNTSNPDITCPPPMS VEHADIWVKS Y SLY SRERYICNSGFKRKAGTS SLT

ECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 130).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA

ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC

AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA

TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG

AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA

CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG

AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG

ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG

CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG

CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA

AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC

CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC

AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA

AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC

GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC

TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG

CGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGC

GTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGA

GGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCAC

CGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCT

TTAAAGTGCATCCGG (SEQ ID NO: 131).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S SAY SIPPHV QKS VNNDMIVTDNNGAVKFPQLCKF CDVRFST

CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMC SC SSDECNDNIIF SEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMC SC S SDECNDNIIF SEEYNT SNPDIT CPPPMS VEHADIWVKS Y SLY SRER YICN S GFKRK AGT S SLTEC VLNK ATN VAHWTTP SLKCIR (SEQ ID NO: 132).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC

AACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT

CAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCC

ATCTGCGAGAAGCCCC AAGAAGT GTGCGT GGCCGTGT GGCGGAAAAAT GAC

GAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG

ACTTC ATTCTGGAGGACGCTGCCTCCCCC AAAT GC AT CAT GAAGGAGAAGAA

GAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAAC

GACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTG

GCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGT

GC AG A AG AGC GT G A AT A AT GAC AT GAT C GT GAC C GAT A AC A AT GGC GC C GT G

AAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA

CCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC

AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA

AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC

GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC

TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG

CGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGC

GTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGA

GGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCAC CGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCT TTAAAGTGCATCCGG (SEQ ID NO: 133).

Exemplary Multi-Chain Chimeric Polypeptides- Type J

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-7, a receptor of IL-21, or a receptor of CD137L. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to a receptor for IL-21 (e.g., a soluble IL-21, e.g., a soluble human IL-21) or a receptor for CD137L (e.g., a soluble CD137L, e.g., a soluble human CD137L).

In some examples of these multi-chain chimeric polypeptides, the first target- binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments, the second chimeric polypeptide can include an additional target-binding domain. In some embodiments, the additional target-binding domain and the

In some embodiments of these multi-chain chimeric polypeptides, one or more of the first target-binding domain, the second target-binding domain and the additional target-binding domain is an agonistic antigen-binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain, the second target-binding domain, and the additional target-binding domain are each agonistic antigen-binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain binds specifically to a receptor for IL-7, and the second target-binding domain binds specifically to a receptor for IL-21 or a receptor for CD137L. In some embodiments, the additional target-binding domain binds specifically to a receptor for IL- 21 or a receptor for CD137L.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain is a soluble IL-7 (e.g., a soluble human IL-7). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM

FLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A ALGE AQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 135).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC

TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA

GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG

AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA

CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA

GC AGAAGAAGCTGAACGACCTGT GCTTCCTGAAGAGGCTGCTGC AGGAGAT C AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO: 197).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain or the additional target-binding domain binds specifically to a receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain or the additional target-binding domain is a soluble IL-21 (e.g., a soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human IL-21 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKS ANTGNNERIINV SIKKLKRKPP STNAGRRQKHRLTCP SCD S YEKKPPKEFLER FK SLLQKMIHQHL S SRTHGSED S (SEQ ID NO: 124).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 125).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain binds specifically to a receptor for CD137L. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises an additional target-binding domain that binds specifically to a receptor for CD137L. In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain and/or the additional target-binding domain is a soluble CD137L (e.g., a soluble human CD137L).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

REGPEL SPDDP AGLLDLRQGMF AQL VAQNVLLIDGPL S WY SDPGL AGV SLTGGL S YKEDTKELVVAK AGVYYVFF QLELRRVVAGEGSGS V SLALHLQPLRS AAGAA AL ALT VDLPP AS SEARN S AF GF QGRLLHL S AGQRLGVHLHTEARARHAW QLT Q GAT VLGLFRVTPEIP AGLP SPRSE (SEQ ID NO: 165).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGC GGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGG GCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGG GGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGA GTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGG CTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTG GGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCT CGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCC AGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCA GCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC CCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 166). In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DP AGLLDLRQGMF AQL VAQNVLLIDGPL S W Y SDPGL AGV SLT GGL S YKEDTKEL WAKAGVYYVFF QLELRRVVAGEGSGS V SLALHLQPLRS AAGAAAL ALTVDLP PAS SE ARN S AFGF QGRLLHLS AGQRLGVHLHTEARARHAW QLTQGAT VLGLFR VTPEI (SEQ ID NO: 167). In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGC ATGTTTGCGCAGCTGGTGG CCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGG CCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAA GGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGC TGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCA CCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGG ACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGC CGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGA GGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGA CTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 168). In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP AALGE AQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAY NLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVK DVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVG TK VN VT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYWK S S S S GKKT AKTNTNE FLID VDKGENY CF S VQ AVIPSRTVNRKSTD SPVECMGQEKGEFRENWVNVISDL KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVEN LIIL ANN SL S SN GN VTE S GCKECEELEEKNIKEFLQ SF VHI V QMFINT S (SEQ ID NO: 141).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG

GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC

TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA

GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG

AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA

CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT

GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA

GC AGAAGAAGCTGAACGACCTGT GCTTCCTGAAGAGGCTGCTGC AGGAGAT C

AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACA

ACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAA

CCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGAT

CAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACC

GAGT GCGATCTC ACCGAT GAGATCGTGAA AGATGT GAAAC AGACCT ACCTCG

CCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGG

CGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATT GACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGC

CTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTC

CTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATC

GACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCT

CCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCA

AGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAA

GAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACA

GAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACT

GGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACC

GTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGT

GAC AGAGTCCGGCTGC A AGGAGT GCGAAGAGCTGGAGGAGAAGAAC AT C A A

GGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

(SEQ ID NO: 142)

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKW VTFISLLFLF S SAY SDCDIEGKDGKQ YES VLMV SIDQLLD SMKEIGSNCLNN EFNFFKRHICD ANKEGMFLFR A ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLN C TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEH S GTTNT V A A YNLT WK S TNFK TILEWEPKP VN Q V YT V QI S TK S GD WK S KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP YLETNLGQPTIQ SFEQ V GTKVNVTVEDERTLVRRNNTFL SLRD VF GKDLI YTL YY WKSS S SGKKTAKTNTNEFLIDVDKGENYCF S VQ A VIP SRT VNRK S TD SP VECMG QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL Q VI SLE S GD ASIHD T VENLIIL ANN SL S SN GN VTE S GCKECEELEEKNIKEFLQ SF V HIVQMFINTS (SEQ ID NO: 143).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGAT

GGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGC

CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG

AGGGC AT GTTCCTGTT C AGGGCCGCC AGGAAACTGCGGC AGTTCCTGAAGAT

GAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACC

ACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC

TGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG

AGC AGAAGAAGCTGAACGACCTGT GCTTCCTGAAGAGGCTGCTGC AGGAGAT

CAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCAC

AACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAA

ACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGA

TCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAC

CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTC

GCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTG

GCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAA

TTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAAT

GTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCA

GCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAG

TCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAA

TCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCC

CTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGC

CAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTA

AAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACA

CAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTA

CTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACA

CCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAAC

GT G AC AG AGT C C GGC T GC A AGG AGT GC G A AG AGC T GG AGG AG A AG A AC ATC AAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTC C (SEQ ID NO: 144)

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKS ANTGNNERIINV SIKKLKRKPP STNAGRRQKHRLTCP SCD S YEKKPPKEFLER FK SLLQKMIHQHL S SRTHGSED SIT CPPPM S VEFLADIW VK S YSL Y SRERYICN S GF KRK AGT S SLTEC VLNK ATN V AHWTTP SLKCIRGGGGS GGGGS GGGGSREGPEL S PDDP AGLLDLRQGMF AQL VAQNVLLIDGPL S W Y SDPGL AGV SLTGGL S YKEDTK ELWAKAGVYYVFF QLELRRVVAGEGSGS V SLALHLQPLRS AAGAAAL ALTVD LPP AS SEARN S AF GF QGRLLHLS AGQRLGVHLHTEARARHAW QLTQGAT VLGLF RVTPEIPAGLPSPRSE (SEQ ID NO: 169).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG

ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC

CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG

GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG

AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG

CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC

CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA

GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTC

CCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAG

CCGGGAGAGGT AT ATCTGT AAC AGCGGCTT C AAGAGGAAGGCCGGC ACC AG CAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACA

ACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCT

CCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGC

CGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAAT

GTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAG

GCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGG

TGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGC

GTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGC

CACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCA

CCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCT

GCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGG

GCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCC

GGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ

ID NO: 170).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEW S AF SCF QKAQLKS ANTGNNERIINV SIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIW VKS Y SLY SRERYICN SGFKRKAGTS SLTEC VLNKATNVAF1WTTPSLKCIRGGGGS GGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPG L AGV SLTGGL S YKEDTKEL V V AKAGV Y Y VFF QLELRRV V AGEGS GS V SL ALHL QPLRS AAGAAAL ALT VDLPP AS SEARN S AF GFQGRLLHL S AGQRLGVHLHTEAR ARH AW QLTQGAT VLGLFRVTPEIP AGLP SPRSE (SEQ ID NO: 171)

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC

GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC

CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA

GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGT

GAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAG

GCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCC

CCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATC

AGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCT

CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA

GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA

GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC

AACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGC

TCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCG

CCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAA

TGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCA

GGCGT GTCCCTGACGGGGGGCCTGAGCT AC AAAGAGGAC ACGAAGGAGCTG

GTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCG

CGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAG

CCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCC

ACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGC

TGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAG

GGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTC

CGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ

ID NO: 172).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKS ANTGNNERIINV SIKKLKRKPP STNAGRRQKHRLTCP SCD S YEKKPPKEFLER FK SLLQKMIHQHL S SRTHGSED SIT CPPPM S VEFLADIW VK S YSL Y SRERYICN S GF KRK AGT S SLTEC VLNK ATN V AHWTTP SLKCIRGGGGS GGGGS GGGGSDP AGLL DLRQGMF AQL VAQNVLLIDGPL S WY SDPGL AGV SLTGGL S YKEDTKEL VVAK A GVYYVFF QLELRRVVAGEGSGS V SLALHLQPLRS AAGAAALALTVDLPP AS SEA RN S AFGF QGRLLHL S AGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEI

(SEQ ID NO: 173).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG

ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC

CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG

GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG

AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG

CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC

CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA

GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTC

CCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAG

CCGGGAGAGGT AT ATCTGT AAC AGCGGCTT C AAGAGGAAGGCCGGC ACC AG

CAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACA

ACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCT

CCGGCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCAT

GTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCT

GGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTA TTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCG

TTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCC

CTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGC

CTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGC

GTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGG

GCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 174).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEW S AF SCF QKAQLKS ANTGNNERIINV SIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIW VKS Y SLY SRERYICN SGFKRKAGTS SLTEC VLNKATNVAF1WTTPSLKCIRGGGGS GGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTG GLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAG AAAL ALT VDLPP AS SE ARN S AFGF QGRLLHL S AGQRLGVHLHTE ARARHAW QL TQGATVLGLFRVTPEI (SEQ ID NO: 175).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

CCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC

GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC

CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA

GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGT

GAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAG GCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCC CCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATC AGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCT CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGC TCCGGCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCA TGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGC TGGTAC AGTGACCC AGGCCTGGC AGGCGTGTCCCTGACGGGGGGCCTGAGCT AC A A AG AGG AC AC G A AGG AGC T GGT GGT GGC C A AGGC T GG AGT CTACTATG TCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTC CGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCG CCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCG GCCTTCGGTTTCCAGGGCCGCTTGCTGC ACCTGAGTGCCGGCCAGCGCCTGG GCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCA GGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 176).

Exemplary Multi-Chain Chimeric Polypeptides- Type K

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-7 or TGF-b. In some examples of these multi-chain chimeric polypeptides, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain binds specifically to a receptor for IL-7, and the second target-binding domain binds specifically to TGF-b. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain binds specifically to TGF-b, and the second target-binding domain binds specifically to a receptor for IL-7.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain includes a soluble IL-7 protein (e.g., a soluble human IL-7 protein). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 protein includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP AALGE AQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 135).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCAC ATCTGCGACGCCAAC AAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GC AGAAGAAGCTGAACGACCTGT GCTTCCTGAAGAGGCTGCTGC AGGAGAT C AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO: 197).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain comprises a target-binding domain that binds specifically to TGF- b. In some embodiments of these multi-chain chimeric polypeptides, the second target- binding domain is a soluble TGF-b receptor(e.g., a soluble TGF-b RII receptor, e.g., a soluble human TGF-b RII receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII includes a first sequence of soluble human TGF-b RII and a second sequence of soluble human TGF-b RII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII includes a linker disposed between the first sequence of soluble human TGF-b RII and the second sequence of soluble human TGF-b RII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO:

7)· In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVC VAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 57) In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGT GAGAAGCCTC AGGAGGT GTGCGT GGCTGTCTGGCGGAAGAAT GACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 57).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-b receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIF SEE YNT SNPD (SEQ ID NO: 60).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-b receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGAC (SEQ ID NO: 198).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKPA ALGE AQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYN LT WK S TNFKTILEWEPKP VN Q V YT V QIS TK S GD WK SKCF YTTDTECDLTDEIVKD VKQTYLARVF S YPAGNVESTGS AGEPLYENSPEFTPYLETNLGQPTIQ SFEQVGTK VN VT VEDERTLVRRNNTFL SLRD VF GKDLI YTLYYWK S S S SGKKTAKTNTNEFLI VECMGQEKGEFRENW VN VI SDLKKIE DLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILA NN SL S SN GN VTE SGCKECEELEEKNIKEFLQ SF VHI V QMFINT S (SEQ ID NO: 141).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATT GC G AC AT C G AGGGC A AGG AC GGC A AGC AGTAC G AG AGC GT GC T GAT GG

TGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTC

AACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGG

GCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAA

CTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACC

ATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGG

GAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGC

AG A AG A AGC T G A AC G AC C T GT GC TT C C T G A AG AGGC T GC T GC AGG AG AT C A A

GACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACC

AACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCAT

CCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCA

CCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGC

GATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGT

GTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTT

TATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAG

CCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGG

AGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGA

TGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTC

CGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGAT

AAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGT

GAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGC

G AGT TC C GGG AG A AC T GGGT G A AC GT CAT C AGC G ATT TA A AG A AG AT C G A AG

ATTTAATT C AGTCC AT GC ATATCGACGCC ACTTTATAC AC AGAATCCGACGT GC

ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTA TCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATC ATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTG CAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCC TTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 142).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN EFNFFKRHICD ANKEGMFLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLN C TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEHS GTTNT VA AYNLTWK STNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SK CFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWK S S S S GKKTAKTNTNEFLID VDKGEN Y CF S VQ AVIP SRT VNRK STD SP VECMGQEK GEFRENW VN VI SDLKKIEDLIQ SMHID ATLYTE SD VHP S CK VTAMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS (SEQ ID NO: 143).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

GATT GC G AC AT C G AGGGC A AGG AC GGC A AGC AGTAC G AG AGC GT GC T GAT GG

TGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTC

AACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGG

GCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAA

CTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACC ATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGG

GAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGC

AG A AG A AGC T G A AC G AC C T GT GC TT C C T G A AG AGGC T GC T GC AGG AG AT C A A

GACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACC

AACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCAT

CCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCA

CCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGC

GATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGT

GTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTT

TATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAG

CCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGG

AGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGA

TGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTC

CGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGAT

AAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGT

GAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGC

G AGT TC C GGG AG A AC T GGGT G A AC GT CAT C AGC G ATT TA A AG A AG AT C G A AG

ATTTAATT C AGTCC AT GC ATATCGACGCC ACTTTATAC AC AGAATCCGACGT GC

ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTA

TCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATC

ATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTG

CAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCC

TTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 144).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE

KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF

FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMC SC S SDECNDNI IFSEEYNTSNPDITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRKAGTS SLTEC VLNKATNVAHWTTPSLKCIR (SEQ ID NO: 130).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG

ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC

AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG

AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG

G (SEQ ID NO: 131). In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKF CDVRFST CDN QK S CM SN C SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPH V QK S VNNDMI VTDNN GAVKFPQLCKF CD VRF S T CDN QK S CM SNC SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKP GETFFMC S C S SDECNDNIIF SEE YNT SNPDIT CPPPMS VEFLADIW VK S Y SLY SRERY ICNSGFKRKAGTS SLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 132).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG

G (SEQ ID NO: 133).

Exemplary Multi-Chain Chimeric Polypeptides- Type L

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to TGF-b, a receptor of IL-21, or a receptor of CD137L. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to a receptor for IL-21 (e.g., a soluble IL-21, e.g., a soluble human IL-21) or a receptor for CD137L (e.g., a soluble CD137L, e.g., a soluble human CD137L).

In some examples of these multi-chain chimeric polypeptides, the first target- binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of the first target-binding domain, the second target-binding domain and the additional target-binding domain is an agonistic antigen-binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain, the second target-binding domain, and the additional target-binding domain are each agonistic antigen-binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain binds specifically to TGF-b and the second target-binding domain binds specifically to a receptor for IL-21 or a receptor for CD137L.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain is a soluble TGF-b receptor (e.g., a soluble TORbKII receptor, e.g., a soluble human TORbKII receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII includes a first sequence of soluble human TGF-b RII and a second sequence of soluble human TGF-b RII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII includes a linker disposed between the first sequence of soluble human TGF-b RII and the second sequence of soluble human TGF-b RII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO:

7)·

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 57)

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA

ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT

TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT

CTGT GAGAAGCCTC AGGAGGT GTGCGT GGCTGTCTGGCGGAAGAAT GACGAG

AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT

CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA

GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 57).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-b receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIF SEE YNT SNPD (SEQ ID NO: 60).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-b receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGAC (SEQ ID NO: 198).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain or the additional target-binding domain binds specifically to a receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain or the additional target-binding domain includes a soluble IL-2l(e.g., a soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human IL-21 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKS ANTGNNERIINV SIKKLKRKPP STNAGRRQKHRLTCP SCD S YEKKPPKEFLER FK SLLQKMIHQHL S SRTHGSED S (SEQ ID NO: 124).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA

GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 125). In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain or the additional target-binding domain binds specifically to a receptor for CD137L. In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain and/or the additional target-binding domain includes a soluble CD137L (e.g., a soluble human CD137L).

In some embodiments of these multi-chain chimeric polypeptides, a soluble CD137L includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

REGPEL SPDDP AGLLDLRQGMF AQL VAQNVLLIDGPL S WY SDPGL AGV SLTGGL S YKEDTKELVVAK AGVYYVFF QLELRRVVAGEGSGS V SLALHLQPLRS AAGAA AL ALT VDLPP AS SEARN S AF GF QGRLLHL S AGQRLGVHLHTEARARHAW QLT Q GAT VLGLFRVTPEIP AGLP SPRSE (SEQ ID NO: 165). In some embodiments of these multi-chain chimeric polypeptides, a soluble

CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGC GGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGG GCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGG GGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGA GTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGG CTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTG GGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCT CGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCC AGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCA GCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC CCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 166). In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L includes a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to:

DP AGLLDLRQGMF AQL VAQNVLLIDGPL S W Y SDPGL AGV SLT GGL S YKEDTKEL WAKAGVYYVFF QLELRRVVAGEGSGS V SLALHLQPLRS AAGAAAL ALTVDLP PAS SE ARN S AFGF QGRLLHLS AGQRLGVHLHTEARARHAW QLTQGAT VLGLFR VTPEI (SEQ ID NO: 167).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to:

GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGG

CCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGG

CCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAA

GGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGC

TGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCA

CCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGG

ACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGC

CGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGA

GGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGA

CTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 168).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE

KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF

FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMC SC S SDECNDNI IF SEEYNT SNPD S GTTNT VA AYNLT WK S TNFKTILEWEPKP VN Q V YT V QI STK S GD WKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPE F TP YLETNLGQPTIQ SFEQ V GTK VN VT VEDERTLVRRNNTFL SLRD VF GKDLI YTL YYWKSS S S GKKTAKTNTNEFLID VDKGEN Y CF S VQ AVIPSRTVNRKSTDSPVECM GQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE LQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSF VHIVQMFINTS (SEQ ID NO: 161).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG

TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC

CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA

AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT

GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT

TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC

AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG

GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT

ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC

AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG

CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC

CC GTT GAGT GC AT GGGC C A AGA A A AGGGC GAGTTCC GGGAGA ACTGGGT GA

ACGT CAT C AGCGATTTAAAGAAGATCGAAGATTTAATT C AGTCC ATGC ATATCG

ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC

ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT

AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC

AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG

GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT

CATCAATACCTCC (SEQ ID NO: 162).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKF CDVRFST CDN QK S CM SN C SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPH V QK S VNNDMI VTDNN GAVKFPQLCKF CD VRF S T CDN QK S CM SNC SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKP GETFFMC SC S SDECNDNIIF SEE YNT SNPD S GTTNT VA AYNLTWK STNFKTILEWE

PKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPA GNVE S T GS AGEPLYEN SPEF TP YLETNLGQPTIQ SFEQ V GTK VN VT VEDERTLVRR NNTFL SLRD VF GKDLI YTLYYWK S S S S GKKTAKTNTNEFLID VDKGEN Y CF S VQ A VIP SRT VNRK STD SP VECMGQEKGEFRENW VN VI SDLKKIEDLIQ SMHID ATLYTE SDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESG CKECEELEEKNIKEFLQ SF VHIV QMFINTS (SEQ ID NO: 163).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC

ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG

TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC

CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT

GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT

TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC

AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG

GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT

ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC

AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG

CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC

CC GTT GAGT GC AT GGGC C A AGA A A AGGGC GAGTTCC GGGAGA ACTGGGT GA

ACGT CAT C AGCGATTTAAAGAAGATCGAAGATTTAATT C AGTCC ATGC ATATCG

ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC

ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT

AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC

AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG

GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT

CATCAATACCTCC (SEQ ID NO: 164).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEW S AF SCF QKAQ LK SANT GNNERIIN V SIKKLKRKPP STN AGRRQKHRLT CP S CD S YEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSED SITCPPPMS VEHADIWVKS Y SLY SRERYICNSGF KRK AGT S SLTEC VLNK ATN VAHWTTP SLKCIRGGGGS GGGGS GGGGSREGPEL SP DDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKE LVVAKAGVYYVFF QLELRRVVAGEGSGS V SLALHLQPLRS AAGAAAL ALTVDLP PAS SEARN S AFGF QGRLLHL S AGQRLGVHLHTEARARHAWQLTQGAT VLGLFRV TPEIPAGLPSPRSE (SEQ ID NO: 169). In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG

ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC

CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG

GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGA

GCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGC

AGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCC

CAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAG

CACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCC

CATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCC

GGGAGAGGTATAT C T GTA AC AGCGGCTT C A AGAGGA AGGCC GGC AC C AGC AG

CCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACA

CCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCG

GCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGG

CCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTC

TGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGT

GT C C C T G AC GGGGGGC C T G AGC TAC A A AG AGG AC AC G A AGG AGC T GGT GGT

GGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGG

TGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACT

GCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCG

CCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCAC

CTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCAC

GCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTG

ACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO:

170)

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEW SAF SCF QKAQLKS ANTGNNERIINV SIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIW VKS Y SLY SRERYICNSGFKRK AGT S SLTEC VLNK ATNVAHWTTP SLKCIRGGGGS GGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPG L AGV SLT GGL S YKEDTKELVVAK AGVYYVFF QLELRRVVAGEGSGS V SL ALHLQ PLRS AAGAAAL ALTVDLPPAS SEARN SAF GF QGRLLHLS AGQRLGVHLHTEARA RH AWQLTQGAT VLGLFRVTPEIPAGLP SPRSE (SEQ ID NO: 171).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG

ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC

CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG

GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGA

GCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGC

AGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCC

CAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAG

CACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCC

CATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCC

GGGAGAGGTATAT C T GTA AC AGCGGCTT C A AGAGGA AGGCC GGC AC C AGC AG

CCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACA

CCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCG

GCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGG

CCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTC TGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGT

GT C C C T G AC GGGGGGC C T G AGC TAC A A AG AGG AC AC G A AGG AGC T GGT GGT

GGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGG

TGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACT

GCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCG

CCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCAC

CTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCAC

GCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTG

ACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO:

172)

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEW S AF SCF QKAQ LK SANT GNNERIIN V SIKKLKRKPP STN AGRRQKHRLT CP S CD S YEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSED SITCPPPMS VEHADIWVKS Y SLY SRERYICNSGF KRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLLD LRQGMFAQLVAQNVLLIDGPL S W Y SDPGL AGV SLTGGL S YKEDTKELVVAK AGV YYVFF QLELRRVVAGEGSGS V SL ALHLQPLRS AAGAAALALTVDLPPAS SEARN S AF GF QGRLLHL S AGQRLGVHLHTE ARARHAWQLTQGATVLGLFRVTPEI (SEQ ID NO: 173).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG

ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG

GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGA

GCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGC

AGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCC

CAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAG

CACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCC

CATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCC

GGGAGAGGTATAT C T GTA AC AGCGGCTT C A AGAGGA AGGCC GGC AC C AGC AG

CCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACA

CCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCG

GCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTT

GCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTA

CAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAA

GAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCT

TTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTC

ACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGG

CTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTC

GGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCC

ATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCC

ACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 174).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEW S AF SCF QKAQLKS ANTGNNERIINV SIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIW VKS Y SLY SRERYICNSGFKRK AGT S SLTEC VLNK ATNVAHWTTP SLKCIRGGGGS GGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPL S W Y SDPGL AGV SLT GG

LSYKEDTKELWAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA AL ALTVDLPPAS SEARN S AF GF QGRLLHL S AGQRLGVHLHTEARARHAWQLTQG ATVLGLFRVTPEI (SEQ ID NO: 175).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG

ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC

CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG

GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGA

GCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGC

AGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCC

CAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAG

CACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCC

CATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCC

GGGAGAGGTATAT C T GTA AC AGCGGCTT C A AGAGGA AGGCC GGC AC C AGC AG

CCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACA

CCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCG

GCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTT

GCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTA

CAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAA

GAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCT

TTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTC

ACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGG

CTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTC

GGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCC

ATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCC

ACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 176). Exemplary Multi-Chain Chimeric Polypeptides- Type M

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to TGF-b or a receptor of IL-21. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to a receptor for IL-21 (e.g., a soluble IL-21, e.g., a soluble human IL-21) or a TGF-b (e.g., a soluble TGF-b receptor, e.g., a soluble TORbIIP receptor).

In some examples of these multi-chain chimeric polypeptides, the first target- binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain binds specifically to TGF-b, and the second target-binding domain binds specifically to TGF-b or a receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain is a soluble TGF-b receptor (e.g., a soluble TORbKII receptor, e.g., a soluble human TORbίIP receptor). In some

embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII includes a first sequence of soluble human TGF-b RII and a second sequence of soluble human TGF-b RII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII includes a linker disposed between the first sequence of soluble human TGF-b RII and the second sequence of soluble human TGF-b RII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGS GGGGS GGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 58).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 57).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCC AGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGT GAGAAGCCTC AGGAGGT GTGCGT GGCTGTCTGGCGGAAGAAT GACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGC AGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO:

59).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-b receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIF SEE YNT SNPD (SEQ ID NO: 60).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-b receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTC AGCGAAGAGTAC AACACCAGC AACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 198).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain binds specifically to a receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain includes a soluble IL-21 (e.g., a human soluble IL-21). In some embodiments of these multi-chain chimeric polypeptides, the soluble IL-21 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKS ANTGNNERIINV SIKKLKRKPP STNAGRRQKHRLTCP SCD S YEKKPPKEFLER FK SLLQKMIHQHL S SRTHGSED S (SEQ ID NO: 124). In some embodiments of these multi-chain chimeric polypeptides, the soluble IL- 21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG

ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC

CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG

GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG

AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG

CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC

CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA

GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 125).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMC SC S SDECNDNI IF SEEYNT SNPD S GTTNT VA AYNLT WK S TNFKTILEWEPKP VN Q V YT V QI STK S GD WKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPE F TP YLETNLGQPTIQ SFEQ V GTK VN VT VEDERTLVRRNNTFL SLRD VF GKDLI YTL YYWKSS S SGKKTAKTNTNEFLID VDKGENYCF S VQ AVIPSRTVNRKSTDSPVECM GQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE LQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSF VHIVQMFINTS (SEQ ID NO: 161). In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC

ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG

TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC

CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA

AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT

GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT

TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC

AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG

GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT

ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG

CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC

CC GTT GAGT GC AT GGGC C A AGA A A AGGGC GAGTTCC GGGAGA ACTGGGT GA

ACGT CAT C AGCGATTTAAAGAAGATCGAAGATTTAATT C AGTCC ATGC ATATCG

ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC

ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT

AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC

AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG

GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT

CATCAATACCTCC (SEQ ID NO: 162).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKF CDVRFST CDN QK S CM SN C SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPH V QK S VNNDMI VTDNN GAVKFPQLCKF CD VRF S T CDN QK S CM SNC SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKP GETFFMC SC S SDECNDNIIF SEE YNT SNPD S GTTNT VA AYNLTWK STNFKTILEWE PKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPA GNVE S T GS AGEPLYEN SPEF TP YLETNLGQPTIQ SFEQ V GTK VN VT VEDERTLVRR NNTFL SLRD VF GKDLI YTLYYWK S S S S GKKTAKTNTNEFLID VDKGEN Y CF S VQ A VIP SRT VNRK STD SP VECMGQEKGEFRENW VN VI SDLKKIEDLIQ SMHID ATLYTE SDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESG CKECEELEEKNIKEFLQ SF VHIV QMFINTS (SEQ ID NO: 163).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC

ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG

TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC

CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA

AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT

GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT

TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC

AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG

GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT

ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC

AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG

CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CC GTT GAGT GC AT GGGC C A AGA A A AGGGC GAGTTCC GGGAGA ACTGGGT GA ACGT CAT C AGCGATTTAAAGAAGATCGAAGATTTAATT C AGTCC ATGC ATATCG ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 164).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMC SC S SDECNDNI IFSEEYNTSNPDITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRKAGTS SLTEC VLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAP EDVETNCEW S AF SCF QKAQLKS ANTGNNERIINV SIKKLKRKPPSTNAGRRQKHR LT CP SCD S YEKKPPKEFLERFKSLLQKMIHQHL S SRTHGSED S (SEQ ID NO: 177).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG

ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC

AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG

AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG

GC AGGGCC AGGAC AGGC AC AT GATCCGGAT GAGGC AGCTC ATCGAC ATCGT C

GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC

CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA

GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG

AGC AT C AAGAAGCTGAAGCGGAAGCCTCCCTCC AC AAACGCCGGC AGGAGG

CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC

CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA

GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 178).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKF CDVRFST CDN QK S CM SN C SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPH V QK S VNNDMI VTDNN GAVKFPQLCKF CD VRF S T CDN QK S CM SNC SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKP GETFFMC S C S SDECNDNIIF SEE YNT SNPDIT CPPPMS VEFLADIW VK S Y SLY SRERY ICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIV DQLKNYVNDLVPEFLPAPEDVETNCEW S AF SCF QKAQLKS ANTGNNERIINV SIK KLKRKPP S TN AGRRQKHRLT CP S CD S YEKKPPKEFLERFK SLL QKMIHQHL S SRT HGSEDS (SEQ ID NO: 179).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG GC AGGGCC AGGAC AGGC AC AT GATCCGGAT GAGGC AGCTC ATCGAC ATCGT C GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGC AT C AAGAAGCTGAAGCGGAAGCCTCCCTCC AC AAACGCCGGC AGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 180).

Exemplary Multi-Chain Chimeric Polypeptides- Type N

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to TGF-b or CD16. In some embodiments of these multi- chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi- chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to CD16 (e.g., an anti-CDl6 scFv) or a TGF-b (e.g., a soluble TGF-b receptor, e.g., a soluble TGE^RII receptor).

In some examples of these multi-chain chimeric polypeptides, the first target- binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain binds specifically to TGF-b, and the second target-binding domain binds specifically to TGF-b or CD16. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain is a soluble TGF-b receptor (e.g., a soluble TORbKII receptor, e.g., a soluble human TORbίIP receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII includes a first sequence of soluble human TGF-b RII and a second sequence of soluble human TGF-b RII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII includes a linker disposed between the first sequence of soluble human TGF-b RII and the second sequence of soluble human TGF-b RII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence

GGGGS GGGGS GGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56). In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCC AAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 57).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGT GAGAAGCCTC AGGAGGT GTGCGT GGCTGTCTGGCGGAAGAAT GACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 57). In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-b receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIF SEE YNT SNPD (SEQ ID NO: 60).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-b receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 198).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain binds specifically to CD 16. In some embodiments of these multi - chain chimeric polypeptides, the second target-binding domain includes an anti-CD 16 scFv. In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a light chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRF S GS S S GNT ASLTIT GAQ AEDE AD Y Y CN SRD S S GNH VVF GGGTKLT V GH

(SEQ ID NO: 149).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a light chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GC AG A AGC C C GGC C AGGC TC C T GT GC T GGT GAT C T AC GGC A AG A AC A AC AGG CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCAT (SEQ ID NO: 150).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a heavy chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINW NGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDY WGQGTLVTVSR (SEQ ID NO: 151).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a heavy chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

G AGGT GC AGC T GGT GG AGT C C GG AGG AGG AGT GGT G AGGC C T GG AGGC T C C

CTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTC

CTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAAC

TGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCA

TCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAG

GGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTC

GACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 152).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMC SC S SDECNDNI IF SEEYNT SNPD S GTTNT VA AYNLT WK S TNFKTILEWEPKP VN Q V YT V QI STK S GD WKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPE F TP YLETNLGQPTIQ SFEQ V GTK VN VT VEDERTLVRRNNTFL SLRD VF GKDLI YTL YYWKSS S S GKKTAKTNTNEFLID VDKGEN Y CF S VQ AVIPSRTVNRKSTDSPVECM GQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE LQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSF VHIVQMFINTS (SEQ ID NO: 161).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC

ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG

TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC

CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA

AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT

GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC

AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG

GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT

ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC

AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG

CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC

CC GTT GAGT GC AT GGGC C A AGA A A AGGGC GAGTTCC GGGAGA ACTGGGT GA

ACGT CAT C AGCGATTTAAAGAAGATCGAAGATTTAATT C AGTCC ATGC ATATCG

ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC

ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT

AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC

AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG

GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT

CATCAATACCTCC (SEQ ID NO: 162).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKF CDVRFST CDN QK S CM SN C SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPH V QK S VNNDMI VTDNN GAVKFPQLCKF CD VRF S T CDN QK S CM SNC SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKP GETFFMC SC S SDECNDNIIF SEE YNT SNPD S GTTNT VA AYNLTWK STNFKTILEWE PKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPA GNVE S T GS AGEPLYEN SPEF TP YLETNLGQPTIQ SFEQ V GTK VN VT VEDERTLVRR NNTFL SLRD VF GKDLI YTLYYWK S S S S GKKTAKTNTNEFLID VDKGEN Y CF S VQ A VIP SRT VNRK STD SP VECMGQEKGEFRENW VN VI SDLKKIEDLIQ SMHID ATLYTE SDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESG

CKECEELEEKNIKEFLQ

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC

ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG

TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC

CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA

AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT

GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT

TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC

AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG

GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC

AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG

CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC

CC GTT GAGT GC AT GGGC C A AGA A A AGGGC GAGTTCC GGGAGA ACTGGGT GA

ACGT CAT C AGCGATTTAAAGAAGATCGAAGATTTAATT C AGTCC ATGC ATATCG

ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC

ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT

AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC

AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG

GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT

CATCAATACCTCC (SEQ ID NO: 164).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMC SC S SDECNDNI IFSEEYNTSNPDITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRKAGTS SLTEC VLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQ QKPGQ AP VLVI YGKNNRP SGIPDRF SGS S SGNTASLTITGAQ AEDEAD YYCNSRD S S GNH VVF GGGTKLT V GHGGGGS GGGGS GGGGSE V QLVES GGGVVRPGGSLRL S CAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDN AKN SLYLQMN SLRAEDTAVYY C ARGRSLLFD YW GQGTLVT V SR (SEQ ID NO: 181).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG

ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC

AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG

AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG

GTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTG

AGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA

GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGG

CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC

CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC

TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA

CCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAG

GAT C C G AGGT GC AGC T GGT GG AGT C C GG AGG AGG AGT GGT G AGGC C T GG AG GCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGC ATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCA TCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTT CACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCC CTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGC TGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 182).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKF CDVRFST CDN QK S CM SN C SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPH V QK S VNNDMI VTDNN GAVKFPQLCKF CD VRF S T CDN QK S CM SNC SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKP GETFFMC S C S SDECNDNIIF SEE YNT SNPDIT CPPPMS VEFLADIW VK S Y SLY SRERY ICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVR ITCQGDSLRS YYASWY QQKPGQ APVLVIY GKNNRPSGIPDRF SGS S SGNTASLTIT GAQ AEDE AD Y Y CN SRD S S GNH VVF GGGTKLT V GHGGGGS GGGGS GGGGSE V Q LVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGG STGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQG TLVTVSR (SEQ ID NO: 183). In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG

ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC

AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG

AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG

GTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTG

AGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA

GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGG

CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC

CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC

TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA

CCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAG

GAT C C G AGGT GC AGC T GGT GG AGT C C GG AGG AGG AGT GGT G AGGC C T GG AG

GCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGC

ATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCA TCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTT CACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCC CTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGC TGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 184).

Exemplary Multi-Chain Chimeric Polypeptides- Type O

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to TGF-b or a receptor of CD137L. In some

embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to a receptor to TGF- b (e.g., a soluble TGF-b receptor, e.g., a soluble TGE^RII receptor) or CD137L.

In some examples of these multi-chain chimeric polypeptides, the first target- binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multi- chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target- binding domain binds specifically to TGF-b, and the second target-binding domain binds specifically to CD137L. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain or the additional target-binding domain is a soluble TGF-b receptor (e.g., a soluble TORbKII receptor, e.g., a soluble human TORbKII receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII includes a first sequence of soluble human TGF-b RII and a second sequence of soluble human TGF-b RII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGF-b RII includes a linker disposed between the first sequence of soluble human TGF-b RII and the second sequence of soluble human TGF-b RII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGS GGGGS GGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMC S C S SDECNDNIIF SEEYNT SNPD (SEQ ID NO: 56).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 57). In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGF-b RII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGT GAGAAGCCTC AGGAGGT GTGCGT GGCTGTCTGGCGGAAGAAT GACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO:

57).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-b receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIF SEE YNT SNPD (SEQ ID NO: 60).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-b receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCC AAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 198). In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain includes a soluble CD137L protein (e.g., a soluble human CD137L protein). In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L includes a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to:

REGPEL SPDDP AGLLDLRQGMF AQL VAQNVLLIDGPL S WY SDPGL AGV SLTGGL S YKEDTKELVVAK AGVYYVFF QLELRRVVAGEGSGS V SLALHLQPLRS AAGAA AL ALT VDLPP AS SEARN S AF GF QGRLLHL S AGQRLGVHLHTEARARHAW QLT Q GAT VLGLFRVTPEIP AGLP SPRSE (SEQ ID NO: 165).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to:

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGC GGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGG GCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGG GGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGA GTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGG CTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTG GGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCT CGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCC AGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCA GCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC CCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 166).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L includes a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to: DP AGLLDLRQGMF AQL VAQNVLLIDGPL S W Y SDPGL AGV SLT GGL S YKEDTKEL WAKAGVYYVFF QLELRRVVAGEGSGS V SLALHLQPLRS AAGAAAL ALTVDLP PAS SE ARN S AFGF QGRLLHLS AGQRLGVHLHTEARARHAW QLTQGAT VLGLFR VTPEI (SEQ ID NO: 167).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to:

GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGG

CCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGG

CCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAA

GGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGC

TGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCA

CCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGG

ACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGC

CGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGA

GGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGA

CTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 168).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE

KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF

FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI

VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN

DENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMC SC S SDECNDNI

IF SEEYNT SNPD S GTTNT VA AYNLT WK S TNFKTILEWEPKP VN Q V YT V QI STK S GD

WKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPE F TP YLETNLGQPTIQ SFEQ V GTK VN VT VEDERTLVRRNNTFL SLRD VF GKDLI YTL YYWKSS S SGKKTAKTNTNEFLID VDKGENYCF S VQ AVIPSRTVNRKSTDSPVECM GQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE LQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSF VHIVQMFINTS (SEQ ID NO: 161).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC

ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG

TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC

CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA

AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT

TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC

AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG

GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT

ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC

AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG

CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC

CC GTT GAGT GC AT GGGC C A AGA A A AGGGC GAGTTCC GGGAGA ACTGGGT GA

ACGT CAT C AGCGATTTAAAGAAGATCGAAGATTTAATT C AGTCC ATGC ATATCG

ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC

ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT

AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC

AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG

GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT

CATCAATACCTCC (SEQ ID NO: 162).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKF CDVRFST CDN QK S CM SN C SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPH V QK S VNNDMI VTDNN GAVKFPQLCKF CD VRF S T CDN QK S CM SNC SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKP GETFFMC SC S SDECNDNIIF SEE YNT SNPD S GTTNT VA AYNLTWK STNFKTILEWE PKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPA GNVE S T GS AGEPLYEN SPEF TP YLETNLGQPTIQ SFEQ V GTK VN VT VEDERTLVRR NNTFL SLRD VF GKDLI YTLYYWK S S S S GKKTAKTNTNEFLID VDKGEN Y CF S VQ A VIP SRT VNRK STD SP VECMGQEKGEFRENW VN VI SDLKKIEDLIQ SMHID ATLYTE SDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESG CKECEELEEKNIKEFLQ SF VHIV QMFINTS (SEQ ID NO: 163).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC

ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG

TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC

CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA

AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT

GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT

TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CC GTT GAGT GC AT GGGC C A AGA A A AGGGC GAGTTCC GGGAGA ACTGGGT GA ACGT CAT C AGCGATTTAAAGAAGATCGAAGATTTAATT C AGTCC ATGC ATATCG ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 164).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMC SC S SDECNDNI IFSEEYNTSNPDITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRKAGTS SLTEC VLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQ GMFAQLVAQNVLLIDGPLSWY SDPGL AGV SLTGGLS YKEDTKELVVAKAGVYYV FF QLELRRVVAGEGSGS V SL ALHLQPLRS AAGAAAL ALT VDLPPAS SEARN S AFG F QGRLLHL S AGQRLGVHLHTE ARARHAWQLTQGAT VLGLFRVTPEIPAGLP SPRS

E (SEQ ID NO: 185). In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG

ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC

AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG

AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG

GGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGA

GGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAG

GGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCT

GAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTG

AGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACT

ATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGC TCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGC

CGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACT

CGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTG

GGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCA

GGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGA

CTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 186).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKF CDVRFST CDN QK S CM SN C SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPH V QK S VNNDMI VTDNN GAVKFPQLCKF CD VRF S T CDN QK S CM SNC SIT SICEKPQE V C VAVWRKNDENITLET V CHDPKLP YHDFILED AASPKCIMKEKKKP GETFFMC S C S SDECNDNIIF SEE YNT SNPDIT CPPPMS VEFLADIW VK S Y SLY SRERY ICN S GFKRK AGT S SLTEC VLNK ATN VAHWTTP SLKCIRGGGGS GGGGS GGGGSRE GPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALAL T VDLPPAS SEARN S AFGF QGRLLHL S AGQRLGVHLHTE ARARHAWQLTQGATVL GLFRVTPEIPAGLP SPRSE (SEQ ID NO: 187).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC ATCCCCCCCCAT GT GC AAAAGAGCGT GAAC AACGATATGATCGT GACCGAC AA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCC AAGAAGT GT GCGT GGCCGT GT GGCGGAA AAAT GACGAGA

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

AT C ATCTT C AGCGAAGAGTAC AAC ACC AGC AACCCTGAT GGAGGT GGCGGATC

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG

ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC

AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG

AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG

GGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGA

GGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAG

GGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCT

GAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTG

AGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACT

ATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGC

TCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGC

CGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACT

CGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTG

GGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCA

GGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGA

CTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 188). Compositions/Kits

Also provided herein are compositions (e.g., pharmaceutical compositions) that include at least one of any of the single-chain chimeric polypeptides, multi-chain chimeric polypeptides, any of the cells, or any of the nucleic acids described herein. In some embodiments, the compositions include at least one of any of the single-chain chimeric polypeptides described herein. In some embodiments, the compositions include at least one of any of the multi-chain chimeric polypeptides described herein. In some embodiments, the compositions include any of the immune cells (e.g., any of the immune cells described herein, e.g., any of the immune cells produced using any of the methods described herein).

In some embodiments, the pharmaceutical compositions are formulated for different routes of administration (e.g., intravenous, subcutaneous). In some

embodiments, the pharmaceutical compositions can include a pharmaceutically acceptable carrier (e.g., phosphate buffered saline).

Single or multiple administrations of pharmaceutical compositions can be given to a subject in need thereof depending on for example: the dosage and frequency as required and tolerated by the subject. The formulation should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms.

Also provided herein are kits that include any of the single-chain chimeric polypeptides, multi-chain chimeric polypeptides, compositions, nucleic acids, or cells (e.g., immune cells) described herein. In some embodiments, the kits can include instructions for performing any of the methods described herein. In some embodiments, the kits can include at least one dose of any of the pharmaceutical compositions described herein.

Also provided herein is an immune cell (e.g., an activated NK cell or T cell) produced by any of the methods described herein. Also provided herein are

pharmaceutical compositions that include any of the activated immune cells (e.g., an activated NK cell or T cell) produced by any of the methods described herein. Also provided herein are kits that include any of the pharmaceutical compositions described herein that include any of the activated immune cells (e.g., an activated NK cell or T cell) produced by any of the methods described herein. Also provided herein are kits that include (i) any of the single-chain chimeric polypeptides described herein, and (ii) any of the IgGl antibody constructs described herein. Also provided herein are kits that include (ii) any of the multi-chain chimeric polypeptides described herein, and (ii) any of the IgGl antibody constructs described herein. In some embodiments of any of the kits described herein, the kits further include instructions for performing any of the methods described herein.

Nucleic Acids/Vectors

Also provided herein are nucleic acids that encode any of the single-chain chimeric polypeptides described herein. Also provided herein are vectors that include any of the nucleic acids encoding any of the single-chain chimeric polypeptides described herein.

Also provided herein are nucleic acids that encode any of the multi-chain chimeric polypeptides described herein. In some embodiments, a first nucleic acid can encode the first chimeric polypeptide and a second nucleic acid can encode the second chimeric polypeptide. In some embodiments, a single nucleic acid can encode both the first chimeric polypeptide and the second chimeric polypeptide.

Also provided herein are vectors that include any of the nucleic acids encoding any of the multi-chain chimeric polypeptides described herein. In some embodiments, a first vector can include a nucleic acid encoding the first chimeric polypeptide and a second vector can include a nucleic acid encoding the second chimeric polypeptide. In some embodiments, a single vector can include a first nucleic acid encoding the first chimeric polypeptide and a second nucleic acid encoding the second chimeric polypeptide.

Also provided herein are nucleic acids that encode any of the IgGl antibody constructs described herein.

Any of the vectors described herein can be an expression vector. For example, an expression vector can include a promoter sequence operably linked to the sequence encoding the single-chain chimeric polypeptide, or the first chimeric polypeptide and the second chimeric polypeptide of a multi-chain chimeric polypeptide. Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vectors (e.g., pSV or pCMV vectors), adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors), and any Gateway® vectors. A vector can, e.g., include sufficient cis-acting elements for expression; other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. Skilled practitioners will be capable of selecting suitable vectors and mammalian cells for making any of the single-chain chimeric polypeptides or multi-chain chimeric polypeptides described herein.

Cells

Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acids described herein that encode any of the single-chain chimeric polypeptides described herein.

Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) that include any of the vectors described herein that encode any of the single-chain chimeric polypeptides described herein.

Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acids described herein that encode any of the multi-chain chimeric polypeptides described herein (e.g., encoding both the first and second chimeric polypeptides). Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acids described herein that encode any of the first chimeric polypeptides described herein. Also provided are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acids described herein that encode any of the second chimeric polypeptides described herein.

Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) that include any of the vectors described herein that encode any of the multi-chain chimeric polypeptides described herein (e.g., encoding both the first and second chimeric polypeptides). Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) that include any of the vectors described herein that encode any of the first chimeric polypeptides described herein. Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) that include any of the vectors described herein that encode any of the second chimeric polypeptides described herein).

In some embodiments of any of the methods described herein, the cell can be a eukaryotic cell. As used herein, the term“eukaryotic cell” refers to a cell having a distinct, membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as mammalian, avian, plant, or insect cells. Non-limiting examples of mammalian cells include Chinese hamster ovary cells and human embryonic kidney cells (e.g., HEK293 cells).

Methods of introducing nucleic acids and expression vectors into a cell (e.g., a eukaryotic cell) are known in the art. Non-limiting examples of methods that can be used to introduce a nucleic acid into a cell include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalefection, hydrodynamic delivery, magnetofection, viral transduction (e.g., adenoviral and lentiviral transduction), and nanoparticle transfection. Methods of Producing Single-Chain and Multi-Chain Chimeric Polypeptides

Also provided herein are methods of producing any of the single-chain chimeric polypeptides described herein that include culturing any of the cells described herein in a culture medium under conditions sufficient to result in the production of the single-chain chimeric polypeptide; and recovering the single-chain chimeric polypeptide from the cell and/or the culture medium.

The recovery of the single-chain chimeric polypeptide from a culture medium or a cell (e.g., a eukaryotic cell) can be performed using techniques well-known in the art (e.g., ammonium sulfate precipitation, polyethylene glycol precipitation, ion-exchange chromatography (anion or cation), chromatography based on hydrophobic interaction, metal-affinity chromatography, ligand-affinity chromatography, and size exclusion chromatography) . Also provided herein are single-chain chimeric polypeptides (e.g., any of the single-chain chimeric polypeptides described herein) produced by any of the methods described herein.

Also provided herein are methods of producing any of the multi-chain chimeric polypeptides described herein that include culturing any of the cells described herein in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide; and recovering the multi-chain chimeric polypeptide from the cell and/or the culture medium.

Also provided herein are method of producing any of the multi-chain chimeric polypeptides described herein that include: culturing any of cells described herein in a first culture medium under conditions sufficient to result in the production of the first chimeric polypeptide; recovering the first chimeric polypeptide from the cell and/or the first culture medium; culturing any of the cells described herein in a second culture medium under conditions sufficient to result in the production of the second chimeric polypeptide; recovering the second chimeric polypeptide from the cell and/or the second culture medium; and combining (e.g., mixing) the recovered first chimeric polypeptide and the recovered second chimeric polypeptide to form the multi-chain chimeric polypeptide (e.g., any of the multi-chain chimeric polypeptides described herein).

The recovery of the multi-chain chimeric polypeptide, the first chimeric polypeptide, or the second chimeric polypeptide from a cell (e.g., a eukaryotic cell) can be performed using techniques well-known in the art (e.g., ammonium sulfate precipitation, polyethylene glycol precipitation, ion-exchange chromatography (anion or cation), chromatography based on hydrophobic interaction, metal-affinity

chromatography, ligand-affinity chromatography, and size exclusion chromatography).

Also provided herein are multi-chain chimeric polypeptides (e.g., any of the multi-chain chimeric polypeptides described herein), first chimeric polypeptides (e.g., any of the first chimeric polypeptides), or second chimeric polypeptides (e.g., any of the second chimeric polypeptides described herein) produced by any of the methods described herein.

Methods of culturing cells are well known in the art. Cells can be maintained in vitro under conditions that favor proliferation, differentiation and growth. Briefly, cells can be cultured by contacting a cell (e.g., any cell) with a cell culture medium that includes the necessary growth factors and supplements to support cell viability and growth.

Methods of Promoting the Activation and Proliferation of an Immune Cell

Also provided herein are methods of promoting the activation and proliferation of an immune cell (e.g., an any of the exemplary immune cells described herein or known in the art) that include contacting an immune cell in a liquid culture medium including an effective amount of (i) any of the single-chain or multi-chain chimeric polypeptides described herein, and (ii) an IgGl antibody construct that comprises at least one antigen binding domain that binds specifically to the linker domain (e.g., a monoclonal or polyclonal human, mouse, rabbit, or goat IgGl antibody that binds specifically to a linker domain or any of the other exemplary IgGl antibody constructs described herein), under conditions that allow for the activation and proliferation of the immune cell.

In some embodiments of these methods, the IgGl antibody construct includes at least one antigen-binding domain that binds specifically to the linker domain. In some embodiments of these methods, the IgGl antibody construct is a monoclonal IgGl antibody, where both of the antigen-binding domains in the monoclonal IgGl antibody bind specifically to the linker domain. In some embodiments of these methods, the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the linker domain.

In some embodiments of these methods, the contacting step is performed for a period of about 2 hours to about 20 days (e.g., about 2 hours to about 18 days, about 2 hours to about 16 days, about 2 hours to about 14 days, about 2 hours to about 12 days, about 2 hours to about 10 days, about 2 hours to about 8 days, about 2 hours to about 7 days, about 2 hours to about 6 days, about 2 hours to about 5 days, about 2 hours to about 4 days, about 2 hours to about 3 days, about 2 hours to about 2 days, about 2 hours to about 1 day, about 6 hours to about 18 days, about 6 hours to about 16 days, about 6 hours to about 14 days, about 6 hours to about 12 days, about 6 hours to about 10 days, about 6 hours to about 8 days, about 6 hours to about 7 days, about 6 hours to about 6 days, about 6 hours to about 5 days, about 6 hours to about 4 days, about 6 hours to about 3 days, about 6 hours to about 2 days, about 6 hours to about 1 day, about 12 hours to about 18 days, about 12 hours to about 16 days, about 12 hours to about 14 days, about 12 hours to about 12 days, about 12 hours to about 10 days, about 12 hours to about 8 days, about 12 hours to about 7 days, about 12 hours to about 6 days, about 12 hours to about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12 hours to about 2 days, about 12 hours to about 1 day, about 1 day to about 18 days, about 1 day to about 16 days, about 1 day to about 15 days, about 1 day to about 14 days, about 1 day to about 12 days, about 1 day to about 10 days, about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 18 days, about 2 days to about 16 days, about 2 days to about 14 days, about 2 days to about 12 days, about 2 days to about 10 days, about 2 days to about 8 days, about 2 days to about 7 days, about 2 days to about 6 days, about 2 days to about 5 days, about 2 days to about 4 days, about 2 days to about 3 days, about 3 days to about 18 days, about 3 days to about 16 days, about 3 days to about 14 days, about 3 days to about 12 days, about 3 days to about 10 days, about 3 days to about 8 days, about 3 days to about 7 days, about 3 days to about 6 days, about 3 days to about 5 days, about 3 days to about 4 days, about 4 days to about 18 days, about 4 days to about 16 days, about 4 days to about 14 days, about 4 days to about 12 days, about 4 days to about 10 days, about 4 days to about 8 days, about 4 days to about 7 days, about 4 days to about 6 days, about 4 days to about 5 days, about 5 days to about 18 days, about 5 days to about 16 days, about 5 days to about 14 days, about 5 days to about 12 days, about 5 days to about 10 days, about 5 days to about 8 days, about 5 days to about 7 days, about 5 days to about 6 days, about 6 days to about 18 days, about 6 days to about 16 days, about 6 days to about 14 days, about 6 days to about 12 days, about 6 days to about 10 days, about 6 days to about 8 days, about 6 days to about 7 days, about 7 days to about 18 days, about 7 days to about 16 days, about 7 days to about 14 days, about 7 days to about 12 days, about 7 days to about 10 days, about 7 days to about 8 days, about 8 days to about 18 days, about 8 days to about 16 days, about 8 days to about 14 days, about 8 days to about 12 days, about 8 days to about 10 days, about 9 days to about 18 days, about 9 days to about 16 days, about 9 days to about 14 days, about 9 days to about 12 days, about 12 days to about 18 days, about 12 days to about 16 days, about 12 days to about 14 days, about 14 days to about 18 days, about 14 days to about 16 days, or about 16 days to about 18 days.

In some embodiments of any of the methods described herein, the liquid culture medium is a serum-free liquid culture medium. In some embodiments of any of the methods described herein, the liquid culture medium is a chemically-defined liquid culture medium.

In some embodiments of any of the methods described herein, the liquid culture medium includes the single-chain or multi-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0.5: 1 to about 2: 1 (e.g., about 0.8: 1 to about 1.2: 1).

In some embodiments of any of the methods described herein, the immune cell is selected from the group consisting of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Thl7 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, gd T cell, an ab T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell. In some embodiments of any of the methods described herein, the immune cell is a T-cell or a natural killer cell.

In some embodiments of any of the methods described herein, the immune cell was previously obtained from a subject. Some embodiments of any of the methods described herein further include obtaining the immune cell from the subject prior to the contacting step.

In some embodiments of any of the methods described herein, the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

Some embodiments of any of the methods described herein further includes, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Some embodiments of any of the methods described herein further include, before the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Some embodiments of any of the methods described herein further include, after the contacting step, isolating the immune cell.

In some embodiments of any of the methods described herein, after the contacting step, the immune cell has an increased level of expression or secretion of one or more proteins selected from the group consisting of: 2B4, CD8, CD1 la, CD 16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, CDl58a,

CD 158b, CDl58e, CD178, CD226, CD253, NKG2A, NKG2C, NKG2D, LIR-l, LILR- Bl, KIR2DL1, KIR3DL1, KIR2DL2, KIR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-l, NKG2A, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF- a, and IFN-g as compared to the level of expression or secretion of the one or more proteins prior to the contacting step.

Some embodiments of any of the methods described herein further include, after the contacting step, administering the immune cell to a subject in need thereof. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having an age-related disease or condition. In some embodiments of any of the methods described herein, the age-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having a cancer. In some embodiments of any of the methods described herein, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some embodiments of any of the methods described herein, the subject has been diagnosed or identified as having an infectious disease. In some embodiments of any of the methods described herein, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Also provided herein is an activated immune cell produced by any of the methods described herein. Also provided herein are pharmaceutical compositions that include any of the immune cells produced by any of the methods described herein. Also provided herein are kits that include any of the pharmaceutical compositions including any of the activated immune cells produced by any of the methods described herein. Methods of Stimulating an Immune Cell

Also provided herein are methods of stimulating an immune cell (e.g., any of the exemplary immune cells described herein or known in the art) that include contacting an immune cell with an effective amount of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein or any of the compositions (e.g., pharmaceutical compositions) described herein. In some examples, the immune cell is contacted in vitro (e.g., in a suitable liquid culture medium under conditions sufficient to result in stimulation of the immune cell). In some examples, the contacting further includes contacting the immune cell with an effective amount of any of the IgGl antibody constructs described herein. In some examples, the immune cell has been previously obtained from a subject (e.g., a mammal, e.g., a human). Some embodiments of these methods further include obtaining the immune cell from the subject prior to the contacting step.

In some examples, the immune cell is contacted in vivo. In such embodiments, the single-chain chimeric polypeptide or multi-chain chimeric polypeptide is

administered to a subject (e.g., a mammal, e.g., a human) in an amount sufficient to result in stimulation of an immune cell in the subject. In some embodiments of any of the methods, compositions, and kits described herein, the IgGl antibody construct (e.g., any of the exemplary IgGl antibody constructs described herein is administered to the subject in combination (e.g., simultaneously or sequentially) with administration of the single- chain chimeric polypeptide or multi-chain chimeric polypeptide (e.g., any of the single- chain chimeric polypeptides or multi-chain chimeric polypeptides described herein).

In some examples of any of the methods described herein, the immune cell can be an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Thl7 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, gd T cell, an ab T cell, a tumor- infiltrating T cell, a CD8 ⁺ T cell, a CD4 ⁺ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, or a natural killer cell, or a combination thereof.

In some examples, the immune cell has previously been genetically-modified to express a chimeric antigen receptor or a recombinant T-cell receptor. In some examples, the immune cell (e.g., any of the immune cells described herein) has previously been genetically-modified to express a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include, after the contacting step, introducing into the immune cell (e.g., any of the immune cells described herein) a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Some embodiments of these methods can further include, after the contacting step, introducing into the immune cell (e.g., any of the immune cells described herein) a nucleic acid encoding a co-stimulatory molecule (e.g., CD28). Some embodiments of these methods can further include administering a therapeutically effective amount of the immune cell to a subject in need thereof (e.g., any of the exemplary subjects described herein).

In some examples, the subject can be a subject identified or diagnosed as having an age-related disease or condition. Non-limiting examples of age-related diseases or disorders include: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples, the subject can be a subject that has been identified or diagnosed as having a cancer. Non-limiting examples of cancers include: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples, the subject can be a subject that has been diagnosed or identified as having an infectious disease. Non-limiting examples of infectious disease include infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, or influenza virus.

Activation of an immune cell can be determined using methods known in the art. For example, activation of an immune cell can be determined by detecting the levels of cytokines, chemokines that are secreted or cytotoxicity granules, activating receptors, and regulatory molecules that are upregulated upon activation of an immune cell. Non limiting examples of cytokines and chemokines that are secreted or cytotoxicity granules, activating receptors, and regulatory molecules that are upregulated upon activation of an immune cell include: IL-2, interferon-g, IL-l, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-l 5, IL-17, IL-18, IL-22, IL-33, leukotriene B4, CCL5, TNFa, perforin, TGFp,

STAT3, STAT4, STAT5, RORyT, FOXP3, GAT A3, granzyme A, granzyme B, 2B4, CD8, CDl la, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, CDl58a, CDl58b, CDl58e, CD178, CD226, CD253, NKG2A, NKG2C, NKG2D, LIR-l, LILR-B1, KIR2DL1, KIR3DL1, KIR2DL2, KIR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-l, NKG2A, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-a, and IFN-g. The detection of these cytokines and chemokines can be performed using an immunoassay (e.g., an enzyme-linked

immunosorbent assay). For example, activation of an immune cell can result in an increase of about 1% to about 800% (e.g., about 1% to about 750%, about 1% to about 700%, about 1% to about 650%, about 1% to about 600%, about 1% to about 550%, about 1% to about 500%, about 1% to about 450%, about 1% to about 400%, about 1% to about 350%, about 1% to about 300%, about 1% to about 280%, about 1% to about 260%, about 1% to about 240%, about 1% to about 220%, about 1% to about 200%, about 1% to about 180%, about 1% to about 160%, about 1% to about 140%, about 1% to about 120%, about 1% to about 100%, about 1% to about 90%, about 1% to about 80%, about 1% to about 70%, about 1% to about 60%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 800%, about 5% to about 750%, about 5% to about 700%, about 5% to about 650%, about 5% to about 600%, about 5% to about 550%, about 5% to about 500%, about 5% to about 450%, about 5% to about 400%, about 5% to about 350%, about 5% to about 300%, about 5% to about 280%, about 5% to about 260%, about 5% to about 240%, about 5% to about 220%, about 5% to about 200%, about 5% to about 180%, about 5% to about 160%, about 5% to about 140%, about 5% to about 120%, about 5% to about 100%, about 5% to about 90%, about 5% to about 80%, about 5% to about 70%, about 5% to about 60%, about 5% to about

50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 800%, about 10% to about 750%, about 10% to about 700%, about 10% to about 650%, about 10% to about 600%, about 10% to about 550%, about 10% to about 500%, about 10% to about 450%, about 10% to about 400%, about 10% to about 350%, about 10% to about 300%, about 10% to about 280%, about 10% to about 260%, about 10% to about 240%, about 10% to about 220%, about 10% to about 200%, about 10% to about 180%, about 10% to about 160%, about 10% to about 140%, about 10% to about 120%, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 800%, about 15% to about 750%, about 15% to about 700%, about 15% to about 650%, about 15% to about 600%, about 15% to about 550%, about 15% to about 500%, about 15% to about 450%, about 15% to about 400%, about 15% to about 350%, about 15% to about 300%, about 15% to about 280%, about 15% to about 260%, about 15% to about 240%, about 15% to about 220%, about 15% to about 200%, about 15% to about 180%, about 15% to about 160%, about 15% to about 140%, about 15% to about 120%, about 15% to about 100%, about 15% to about 90%, about 15% to about 80%, about 15% to about 70%, about 15% to about 60%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 800%, about 20% to about 750%, about 20% to about 700%, about 20% to about 650%, about 20% to about 600%, about 20% to about 550%, about 20% to about 500%, about 20% to about 450%, about 20% to about 400%, about 20% to about 350%, about 20% to about 300%, about 20% to about 280%, about 20% to about 260%, about 20% to about 240%, about 20% to about 220%, about 20% to about 200%, about 20% to about 180%, about 20% to about 160%, about 20% to about 140%, about 20% to about 120%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 800%, about 25% to about 750%, about 25% to about 700%, about 25% to about 650%, about 25% to about 600%, about 25% to about 550%, about 25% to about 500%, about 25% to about 450%, about 25% to about 400%, about 25% to about 350%, about 25% to about 300%, about 25% to about 280%, about 25% to about 260%, about 25% to about 240%, about 25% to about 220%, about 25% to about 200%, about 25% to about 180%, about 25% to about 160%, about 25% to about 140%, about 25% to about 120%, about 25% to about 100%, about 25% to about 90%, about 25% to about 80%, about 25% to about 70%, about 25% to about 60%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 35% to about 800%, about 35% to about 750%, about 35% to about

700%, about 35% to about 650%, about 35% to about 600%, about 35% to about 550%, about 35% to about 500%, about 35% to about 450%, about 35% to about 400%, about 35% to about 350%, about 35% to about 300%, about 35% to about 280%, about 35% to about 260%, about 35% to about 240%, about 35% to about 220%, about 35% to about 200%, about 35% to about 180%, about 35% to about 160%, about 35% to about 140%, about 35% to about 120%, about 35% to about 100%, about 35% to about 90%, about 35% to about 80%, about 35% to about 70%, about 35% to about 60%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 800%, about 40% to about 750%, about 40% to about 700%, about 40% to about 650%, about 40% to about 600%, about 40% to about 550%, about 40% to about 500%, about 40% to about 450%, about 40% to about 400%, about 40% to about 350%, about 40% to about 300%, about 40% to about 280%, about 40% to about 260%, about 40% to about 240%, about 40% to about 220%, about 40% to about 200%, about 40% to about 180%, about 40% to about 160%, about 40% to about 140%, about 40% to about 120%, about 40% to about 100%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 40% to about 45%, about 45% to about 800%, about 45% to about 750%, about 45% to about 700%, about 45% to about 650%, about 45% to about 600%, about 45% to about 550%, about 45% to about 500%, about 45% to about 450%, about 45% to about 400%, about 45% to about 350%, about 45% to about 300%, about 45% to about 280%, about 45% to about 260%, about 45% to about 240%, about 45% to about 220%, about 45% to about 200%, about 45% to about 180%, about 45% to about 160%, about 45% to about 140%, about 45% to about 120%, about 45% to about 100%, about 45% to about 90%, about 45% to about 80%, about 45% to about 70%, about 45% to about 60%, about 45% to about 50%, about 50% to about 800%, about 50% to about 750%, about 50% to about 700%, about 50% to about 650%, about 50% to about 600%, about 50% to about 550%, about 50% to about 500%, about 50% to about 450%, about 50% to about 400%, about 50% to about 350%, about 50% to about 300%, about 50% to about 280%, about 50% to about 260%, about 50% to about 240%, about 50% to about 220%, about 50% to about 200%, about 50% to about 180%, about 50% to about 160%, about 50% to about 140%, about 50% to about 120%, about 50% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 800%, about 60% to about 750%, about 60% to about 700%, about 60% to about 650%, about 60% to about 600%, about 60% to about 550%, about 60% to about 500%, about 60% to about 450%, about 60% to about 400%, about 60% to about 350%, about 60% to about 300%, about 60% to about 280%, about 60% to about 260%, about 60% to about 240%, about 60% to about 220%, about 60% to about 200%, about 60% to about 180%, about 60% to about 160%, about 60% to about 140%, about 60% to about 120%, about 60% to about 100%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 800%, about 70% to about 750%, about 70% to about 700%, about 70% to about 650%, about 70% to about 600%, about 70% to about 550%, about 70% to about 500%, about 70% to about 450%, about 70% to about 400%, about 70% to about 350%, about 70% to about 300%, about 70% to about 280%, about 70% to about 260%, about 70% to about 240%, about 70% to about 220%, about 70% to about 200%, about 70% to about 180%, about 70% to about 160%, about 70% to about 140%, about 70% to about 120%, about 70% to about 100%, about 70% to about 90%, about 70% to about 80%, about 80% to about 800%, about 80% to about 750%, about 80% to about 700%, about 80% to about 650%, about 80% to about 600%, about 80% to about 550%, about 80% to about 500%, about 80% to about 450%, about 80% to about 400%, about 80% to about 350%, about 80% to about 300%, about 80% to about 280%, about 80% to about 260%, about 80% to about 240%, about 80% to about 220%, about 80% to about 200%, about 80% to about 180%, about 80% to about 160%, about 80% to about 140%, about 80% to about 120%, about 80% to about 100%, about 80% to about 90%, about 90% to about 800%, about 90% to about 750%, about 90% to about 700%, about 90% to about 650%, about 90% to about 600%, about 90% to about 550%, about 90% to about 500%, about 90% to about 450%, about 90% to about 400%, about 90% to about 350%, about 90% to about 300%, about 90% to about 280%, about 90% to about 260%, about 90% to about 240%, about 90% to about 220%, about 90% to about 200%, about 90% to about 180%, about 90% to about 160%, about 90% to about 140%, about 90% to about 120%, about 90% to about 100%, about 100% to about 800%, about 100% to about 750%, about 100% to about

700%, about 100% to about 650%, about 100% to about 600%, about 100% to about 550%, about 100% to about 500%, about 100% to about 450%, about 100% to about

400%, about 100% to about 350%, about 100% to about 300%, about 100% to about

280%, about 100% to about 260%, about 100% to about 240%, about 100% to about

220%, about 100% to about 200%, about 100% to about 180%, about 100% to about

160%, about 100% to about 140%, about 100% to about 120%, about 120% to about 800%, about 120% to about 750%, about 120% to about 700%, about 120% to about

650%, about 120% to about 600%, about 120% to about 550%, about 120% to about

500%, about 120% to about 450%, about 120% to about 400%, about 120% to about

350%, about 120% to about 300%, about 120% to about 280%, about 120% to about

260%, about 120% to about 240%, about 120% to about 220%, about 120% to about 200%, about 120% to about 180%, about 120% to about 160%, about 120% to about

140%, about 140% to about 800%, about 140% to about 750%, about 140% to about

700%, about 140% to about 650%, about 140% to about 600%, about 140% to about

550%, about 140% to about 500%, about 140% to about 450%, about 140% to about

400%, about 140% to about 350%, about 140% to about 300%, about 140% to about 280%, about 140% to about 260%, about 140% to about 240%, about 140% to about

220%, about 140% to about 200%, about 140% to about 180%, about 140% to about 160%, about 160% to about 800%, about 160% to about 750%, about 160% to about 700%, about 160% to about 650%, about 160% to about 600%, about 160% to about 550%, about 160% to about 500%, about 160% to about 450%, about 160% to about 400%, about 160% to about 350%, about 160% to about 300%, about 160% to about 280%, about 160% to about 260%, about 160% to about 240%, about 160% to about

220%, about 160% to about 200%, about 160% to about 180%, about 180% to about 800%, about 180% to about 750%, about 180% to about 700%, about 180% to about 650%, about 180% to about 600%, about 180% to about 550%, about 180% to about 500%, about 180% to about 450%, about 180% to about 400%, about 180% to about 350%, about 180% to about 300%, about 180% to about 280%, about 180% to about

260%, about 180% to about 240%, about 180% to about 220%, about 180% to about 200%, about 200% to about 800%, about 200% to about 750%, about 200% to about 700%, about 200% to about 650%, about 200% to about 600%, about 200% to about 550%, about 200% to about 500%, about 200% to about 450%, about 200% to about 400%, about 200% to about 350%, about 200% to about 300%, about 200% to about

280%, about 200% to about 260%, about 200% to about 240%, about 200% to about 220%, about 220% to about 800%, about 220% to about 750%, about 220% to about 700%, about 220% to about 650%, about 220% to about 600%, about 220% to about 550%, about 220% to about 500%, about 220% to about 450%, about 220% to about 400%, about 220% to about 350%, about 220% to about 300%, about 220% to about

280%, about 220% to about 260%, about 220% to about 240%, about 240% to about 800%, about 240% to about 750%, about 240% to about 700%, about 240% to about 650%, about 240% to about 600%, about 240% to about 550%, about 240% to about 500%, about 240% to about 450%, about 240% to about 400%, about 240% to about 350%, about 240% to about 300%, about 240% to about 280%, about 240% to about

260%, about 260% to about 800%, about 260% to about 750%, about 260% to about 700%, about 260% to about 650%, about 260% to about 600%, about 260% to about 550%, about 260% to about 500%, about 260% to about 450%, about 260% to about 400%, about 260% to about 350%, about 260% to about 300%, about 260% to about 280%, about 280% to about 800%, about 280% to about 750%, about 280% to about

700%, about 280% to about 650%, about 280% to about 600%, about 280% to about 550%, about 280% to about 500%, about 280% to about 450%, about 280% to about 400%, about 280% to about 350%, about 280% to about 300%, about 300% to about 800%, about 300% to about 750%, about 300% to about 700%, about 300% to about 650%, about 300% to about 600%, about 300% to about 550%, about 300% to about 500%, about 300% to about 450%, about 300% to about 400%, about 300% to about 350%, about 350% to about 800%, about 350% to about 750%, about 350% to about 700%, about 350% to about 650%, about 350% to about 600%, about 350% to about 550%, about 350% to about 500%, about 350% to about 450%, about 350% to about 400%, about 400% to about 800%, about 400% to about 750%, about 400% to about 700%, about 400% to about 650%, about 400% to about 600%, about 400% to about 550%, about 400% to about 500%, about 400% to about 450%, about 450% to about 800%, about 450% to about 750%, about 450% to about 700%, about 450% to about 650%, about 450% to about 600%, about 450% to about 550%, about 450% to about 500%, about 500% to about 800%, about 500% to about 750%, about 500% to about 700%, about 500% to about 650%, about 500% to about 600%, about 500% to about 550%, about 550% to about 800%, about 550% to about 750%, about 550% to about 700%, about 550% to about 650%, about 550% to about 600%, about 600% to about 800%, about 600% to about 750%, about 600% to about 700%, about 600% to about 650%, about 650% to about 800%, about 650% to about 750%, about 650% to about 700%, about 700% to about 800%, about 700% to about 750%, or about 750% to about

800%) of one or more of any of the cytokines, chemokines, cytotoxicity granules, activating receptors or regulatory molecules described herein (e.g., one or more of any of

IL-2, interferon-g, IL-l, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-22, IL-33, leukotriene B4, CCL5, TNFa, granzymes, perforin, TGFP, STAT3,

STAT4, STAT5, RORyT, FOXP3, and GATA3) (e.g., as compared to the level of the one or more cytokines and chemokines in a control not contacted with any of the single- chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein). Methods of Inducing or Increasing Proliferation of an Immune Cell

Also provided herein are methods of inducing or increasing proliferation of an immune cell (e.g., any of the exemplary immune cells described herein or known in the art) that include contacting an immune cell with an effective amount of any of the single- chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein or any of the compositions (e.g., pharmaceutical compositions) described herein.

In some examples, the immune cell is contacted in vitro (e.g., in a suitable liquid culture medium under conditions sufficient to result in stimulation of the immune cell)). In some examples, the immune cell is further contacted in vitro with any of the IgGl antibody constructs described herein in combination (e.g., simultaneously or sequentially) with contacting the immune cell in vitro with the single-chain chimeric polypeptide or multi- chain chimeric polypeptide (e.g., any of the single-chain chimeric polypeptides or multi- chain chimeric polypeptides described herein).

In some examples, the immune cell has been previously obtained from a subject (e.g., a mammal, e.g., a human). Some embodiments of these methods further include obtaining the immune cell from the subject prior to the contacting step.

In some examples, the immune cell is contacted in vivo. In such embodiments, the single-chain chimeric polypeptide or multi-chain chimeric polypeptide is

administered to a subject (e.g., a mammal, e.g., a human) in an amount sufficient to result in stimulation of an immune cell in the subject. In such embodiments, the subject can further be administered any of the IgGl antibody constructs described herein.

In some examples of any of the methods described herein, the immune cell can be an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Thl7 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, gd T cell, an ab T cell, a tumor- infiltrating T cell, a CD8 ⁺ T cell, a CD4 ⁺ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, or a natural killer cell, or a combination thereof.

In some examples, the immune cell has previously been genetically-modified to express a chimeric antigen receptor or a recombinant T-cell receptor. In some examples, the immune cell (e.g., any of the immune cells described herein) has previously been genetically-modified to express a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include, after the contacting step, introducing into the immune cell (e.g., any of the immune cells described herein) a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Some embodiments of these methods can further include, after the contacting step, introducing into the immune cell (e.g., any of the immune cells described herein) a nucleic acid encoding a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include administering a therapeutically effective amount of the immune cell to a subject in need thereof (e.g., any of the exemplary subjects described herein).

In some examples, the subject can be a subject identified or diagnosed as having an age-related disease or condition. Non-limiting examples of age-related diseases or disorders include: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples, the subject can be a subject that has been identified or diagnosed as having a cancer. Non-limiting examples of cancers include: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples, the subject can be a subject that has been diagnosed or identified as having an infectious disease. Non-limiting examples of infectious disease include infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, or influenza virus.

Detection of the proliferation of an immune cell can be performed using methods known in the art, e.g., cytometry (e.g., fluorescence-assisted flow cytometry), microscopy, and immunofluorescence microscopy, e.g., by comparing the rate of increase in the concentration of the immune cell in a sample not contacted with a single-chain chimeric polypeptide or a multi-chain chimeric polypeptide to the rate of increase in the concentration of the immune cell in a similar sample contacted with any of the single chain chimeric polypeptides described herein or any of the multi-chain chimeric polypeptides described herein).

In other examples, the proliferation of an immune cell can be indirectly detected by detecting an increase in the level of one or more cytokines or chemokines secreted or cytotoxicity granules, activating receptors, regulatory molecules upregulated by proliferating immune cells (e.g., one or more of IL-2, IFN-g, IL-l, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-22, IL-33, leukotriene B4, CCL5, TNFa, perforin, TGFp, STAT3, RORyT, FOXP3, STAT6, GAT A3, granzyme A, granzyme B, 2B4, CD8, CDl la, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, CDl58a, CDl58b, CDl58e, CD178,

CD226, CD253, NKG2A, NKG2C, NKG2D, LIR-l, LILR-B1, KIR2DL1, KIR3DL1, KIR2DL2, KIR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-l, NKG2A, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-a, and IFN-g) (e.g, as compared to the level of the one or more cytokines and chemokines in a control not contacted with any of the single-chain chimeric polypeptides described herein). In some embodiments, the methods provided herein can result in an increase (e.g., about 1% to about 800% increase, or any of the subranges of this range described herein) in the rate of increase in the concentration of the immune cell in a sample contacted with any of the single-chain chimeric polypeptides any of the multi-chain chimeric

polypeptides described herein as compared to the rate of increase in a similar control sample not contacted with any of the single-chain chimeric polypeptides any of the multi- chain chimeric polypeptides described herein.

Methods of Inducing Differentiation of an Immune Cell

Also provided herein are method of inducing differentiation of an immune cell (e.g., any of the exemplary immune cells described herein or known in the art) into a memory or memory-like immune cell that include contacting an immune cell with an effective amount of any of the single-chain chimeric polypeptides or any of the multi- chain chimeric polypeptides described herein or any of the compositions (e.g., pharmaceutical compositions) described herein. In some examples, the immune cell is contacted in vitro (e.g., in a suitable liquid culture medium under conditions sufficient to result in stimulation of the immune cell). In some examples, the immune cell is further contacted in vitro with an effective amount of any of the IgGl antibody constructs described herein.

In some examples, the immune cell has been previously obtained from a subject (e.g., a mammal, e.g., a human). Some embodiments of these methods further include obtaining the immune cell from the subject prior to the contacting step.

In some examples, the immune cell is contacted in vivo. In such embodiments, the single-chain chimeric polypeptide or multi-chain chimeric polypeptide is

administered to a subject (e.g., a mammal, e.g., a human) in an amount sufficient to result in stimulation of an immune cell in the subject. In some examples, the immune cell is contacted in vivo with any of the IgGl antibody constructs described herein in

combination (e.g., simultaneously or sequentially) with contacting the immune cell in vivo with the single-chain chimeric polypeptide or multi-chain chimeric polypeptide (e.g., any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein). In some examples of any of the methods described herein, the immune cell can be an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a Thl7 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, gd T cell, an ab T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, or a natural killer cell, or a combination thereof.

In some examples, the immune cell has previously been genetically-modified to express a chimeric antigen receptor or a recombinant T-cell receptor. In some examples, the immune cell (e.g., any of the immune cells described herein) has previously been genetically-modified to express a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include, after the contacting step, introducing into the immune cell (e.g., any of the immune cells described herein) a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Some embodiments of these methods can further include, after the contacting step, introducing into the immune cell (e.g., any of the immune cells described herein) a nucleic acid encoding a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include administering a therapeutically effective amount of the immune cell to a subject in need thereof (e.g., any of the exemplary subjects described herein).

In some examples, the subject can be a subject identified or diagnosed as having an age-related disease or condition. Non-limiting examples of age-related diseases or disorders include: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples, the subject can be a subject that has been identified or diagnosed as having a cancer. Non-limiting examples of cancers include: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples, the subject can be a subject that has been diagnosed or identified as having an infectious disease. Non-limiting examples of infectious disease include infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, or influenza virus.

In some examples, the immune cell is a NK cell, and the detection of a memory NK cell can include, e.g., the detection of the level of one or more of IL-12, IL-18, IL-33, STAT4, Zbtb32, DNAM-l, BIM, Noxa, SOCS1, BNIP3, BNIP3L, IFN-g, TNFa, CXCL16, CXCR6, NKG2D, TRAIL, CD49, CD25, CD69, CD62L, Ly49D, CD49b, and Ly79H. A description of NK memory cells and methods of detecting the same is described in O’Sullivan et ah, Immunity 43:634-645, 2015.

In some examples, the immune cell is a T cell, and the detection of memory T cells can include, e.g., the detection of the level of expression of one or more of

CD45RO, CCR7, L-selectin (CD62L), CD44, CD45RA, integrin aeb7, CD43, CD27, CD28, IL-7Ra, CD95, IL-2Ra, CXCR3, and LFA-l. In some examples, the immune cell is a B cell and the detection of memory B cells can include, e.g., the detection of the level of expression of CD27. Other types and markers of memory or memory-like immune cells are known in the art.

Methods of Treatment

Also provided herein are methods of treating a subject in need thereof (e.g., any of the exemplary subjects described herein or known in the art) that include administering to the subject a therapeutically effective amount of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein or any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, a subject can further be administered a therapeutically effective amount of any of the IgGl antibody constructs described herein.

In some embodiments of these methods, the subject has been identified or diagnosed as having a cancer. Non-limiting examples of cancer include: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of the cancer in the subject (e.g., as compared to the number, severity, or frequency of the one or more symptoms of the cancer in the subject prior to treatment). In some embodiments, these methods can result in a reduction (e.g., about 1% reduction to about 99% reduction, about 1% reduction to about 95% reduction, about 1% reduction to about 90% reduction, about 1% reduction to about 85% reduction, about 1% reduction to about 80% reduction, about 1% reduction to about 75% reduction, about 1% reduction to about 70% reduction, about 1% reduction to about 65% reduction, about 1% reduction to about 60% reduction, about 1% reduction to about 55% reduction, about 1% reduction to about 50% reduction, about 1% reduction to about 45% reduction, about 1% reduction to about 40% reduction, about 1% reduction to about 35% reduction, about 1% reduction to about 30% reduction, about 1% reduction to about 25% reduction, about 1% reduction to about 20% reduction, about 1% reduction to about 15% reduction, about 1% reduction to about 10% reduction, about 1% reduction to about 5% reduction, about 5% reduction to about 99% reduction, about 5% reduction to about 95% reduction, about 5% reduction to about 90% reduction, about 5% reduction to about 85% reduction, about 5% reduction to about 80% reduction, about 5% reduction to about 75% reduction, about 5% reduction to about 70% reduction, about 5% reduction to about 65% reduction, about 5% reduction to about 60% reduction, about 5% reduction to about 55% reduction, about 5% reduction to about 50% reduction, about 5% reduction to about 45% reduction, about 5% reduction to about 40% reduction, about 5% reduction to about 35% reduction, about 5% reduction to about 30% reduction, about 5% reduction to about 25% reduction, about 5% reduction to about 20% reduction, about 5% reduction to about 15% reduction, about 5% reduction to about 10% reduction, about 10% reduction to about 99% reduction, about 10% reduction to about 95% reduction, about 10% reduction to about 90% reduction, about 10% reduction to about 85% reduction, about 10% reduction to about 80% reduction, about 10% reduction to about 75% reduction, about 10% reduction to about 70% reduction, about 10% reduction to about 65% reduction, about 10% reduction to about 60% reduction, about 10% reduction to about 55% reduction, about 10% reduction to about 50% reduction, about 10% reduction to about 45% reduction, about 10% reduction to about 40% reduction, about 10% reduction to about 35% reduction, about 10% reduction to about 30% reduction, about 10% reduction to about 25% reduction, about 10% reduction to about 20% reduction, about 10% reduction to about 15% reduction, about 15% reduction to about 99% reduction, about 15% reduction to about 95% reduction, about 15% reduction to about 90% reduction, about 15% reduction to about 85% reduction, about 15% reduction to about 80% reduction, about 15% reduction to about 75% reduction, about 15% reduction to about 70% reduction, about 15% reduction to about 65% reduction, about 15% reduction to about 60% reduction, about 15% reduction to about 55% reduction, about 15% reduction to about 50% reduction, about 15% reduction to about 45% reduction, about 15% reduction to about 40% reduction, about 15% reduction to about 35% reduction, about 15% reduction to about 30% reduction, about 15% reduction to about 25% reduction, about 15% reduction to about 20% reduction, about 20% reduction to about 99% reduction, about 20% reduction to about 95% reduction, about 20% reduction to about 90% reduction, about 20% reduction to about 85% reduction, about 20% reduction to about 80% reduction, about 20% reduction to about 75% reduction, about 20% reduction to about 70% reduction, about 20% reduction to about 65% reduction, about 20% reduction to about 60% reduction, about 20% reduction to about 55% reduction, about 20% reduction to about 50% reduction, about 20% reduction to about 45% reduction, about 20% reduction to about 40% reduction, about 20% reduction to about 35% reduction, about 20% reduction to about 30% reduction, about 20% reduction to about 25% reduction, about 25% reduction to about 99% reduction, about 25% reduction to about 95% reduction, about 25% reduction to about 90% reduction, about 25% reduction to about 85% reduction, about 25% reduction to about 80% reduction, about 25% reduction to about 75% reduction, about 25% reduction to about 70% reduction, about 25% reduction to about 65% reduction, about 25% reduction to about 60% reduction, about 25% reduction to about 55% reduction, about 25% reduction to about 50% reduction, about 25% reduction to about 45% reduction, about 25% reduction to about 40% reduction, about 25% reduction to about 35% reduction, about 25% reduction to about 30% reduction, about 30% reduction to about 99% reduction, about 30% reduction to about 95% reduction, about 30% reduction to about 90% reduction, about 30% reduction to about 85% reduction, about 30% reduction to about 80% reduction, about 30% reduction to about 75% reduction, about 30% reduction to about 70% reduction, about 30% reduction to about 65% reduction, about 30% reduction to about 60% reduction, about 30% reduction to about 55% reduction, about 30% reduction to about 50% reduction, about 30% reduction to about 45% reduction, about 30% reduction to about 40% reduction, about 30% reduction to about 35% reduction, about 35% reduction to about 99% reduction, about 35% reduction to about 95% reduction, about 35% reduction to about 90% reduction, about 35% reduction to about 85% reduction, about 35% reduction to about 80% reduction, about 35% reduction to about 75% reduction, about 35% reduction to about 70% reduction, about 35% reduction to about 65% reduction, about 35% reduction to about 60% reduction, about 35% reduction to about 55% reduction, about 35% reduction to about 50% reduction, about 35% reduction to about 45% reduction, about 35% reduction to about 40% reduction, about 40% reduction to about 99% reduction, about 40% reduction to about 95% reduction, about 40% reduction to about 90% reduction, about 40% reduction to about 85% reduction, about 40% reduction to about 80% reduction, about 40% reduction to about 75% reduction, about 40% reduction to about 70% reduction, about 40% reduction to about 65% reduction, about 40% reduction to about 60% reduction, about 40% reduction to about 55% reduction, about 40% reduction to about 50% reduction, about 40% reduction to about 45% reduction, about 45% reduction to about 99% reduction, about 45% reduction to about 95% reduction, about 45% reduction to about 90% reduction, about 45% reduction to about 85% reduction, about 45% reduction to about 80% reduction, about 45% reduction to about 75% reduction, about 45% reduction to about 70% reduction, about 45% reduction to about 65% reduction, about 45% reduction to about 60% reduction, about 45% reduction to about 55% reduction, about 45% reduction to about 50% reduction, about 50% reduction to about 99% reduction, about 50% reduction to about 95% reduction, about 50% reduction to about 90% reduction, about 50% reduction to about 85% reduction, about 50% reduction to about 80% reduction, about 50% reduction to about 75% reduction, about 50% reduction to about 70% reduction, about 50% reduction to about 65% reduction, about 50% reduction to about 60% reduction, about 50% reduction to about 55% reduction, about 55% reduction to about 99% reduction, about 55% reduction to about 95% reduction, about 55% reduction to about 90% reduction, about 55% reduction to about 85% reduction, about 55% reduction to about 80% reduction, about 55% reduction to about 75% reduction, about 55% reduction to about 70% reduction, about 55% reduction to about 65% reduction, about 55% reduction to about 60% reduction, about 60% reduction to about 99% reduction, about 60% reduction to about 95% reduction, about 60% reduction to about 90% reduction, about 60% reduction to about 85% reduction, about 60% reduction to about 80% reduction, about 60% reduction to about 75% reduction, about 60% reduction to about 70% reduction, about 60% reduction to about 65% reduction, about 65% reduction to about 99% reduction, about 65% reduction to about 95% reduction, about 65% reduction to about 90% reduction, about 65% reduction to about 85% reduction, about 65% reduction to about 80% reduction, about 65% reduction to about 75% reduction, about 65% reduction to about 70% reduction, about 70% reduction to about 99% reduction, about 70% reduction to about 95% reduction, about 70% reduction to about 90% reduction, about 70% reduction to about 85% reduction, about 70% reduction to about 80% reduction, about 70% reduction to about 75% reduction, about 75% reduction to about 99% reduction, about 75% reduction to about 95% reduction, about 75% reduction to about 90% reduction, about 75% reduction to about 85% reduction, about 75% reduction to about 80% reduction, about 80% reduction to about 99% reduction, about 80% reduction to about 95% reduction, about 80% reduction to about 90% reduction, about 80% reduction to about 85% reduction, about 85% reduction to about 99% reduction, about 85% reduction to about 95% reduction, about 85% reduction to about 90% reduction, about 90% reduction to about 99% reduction, about 90% reduction to about 95% reduction, or about 95% reduction to about 99% reduction) in the volume of one or more solid tumors in the subject (e.g., as compared to the volume of the one or more solid tumors prior to treatment or at the start of treatment). In some embodiments, the these methods can reduce (e.g., about 1% reduction to about 99% reduction, or any of the subranges of this range described herein) the risk of developing a metastasis or developing one or more additional metastasis in a subject (e.g., as compared to the risk of developing a metastasis or developing one or more additional metastasis in a subject prior to treatment or in a similar subject or a population of subjects administered a different treatment).

In some examples of these methods, the subject has been identified or diagnosed as having an aging-related disease or condition. Non-limiting examples of aging-related diseases and conditions include Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some examples, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of the aging- related disease or condition in the subject (e.g., as compared to the number, severity, or frequency of the one or more symptoms of the aging-related disease or condition in the subject prior to treatment). In some examples, the methods can result in a decrease (e.g., about 1% decrease to about 99% decrease, an about 1% decrease to about 95% decrease, about 1% decrease to about 90% decrease, about 1% decrease to about 85% decrease, about 1% decrease to about 80% decrease, about 1% decrease to about 75% decrease, about 1% to about 70% decrease, about 1% decrease to about 65% decrease, about 1% decrease to about 60% decrease, about 1% decrease to about 55% decrease, about 1% decrease to about 50% decrease, about 1% decrease to about 45% decrease, about 1% decrease to about 40% decrease, about 1% decrease to about 35% decrease, about 1% decrease to about 30% decrease, about 1% decrease to about 25% decrease, about 1% decrease to about 20% decrease, about 1% decrease to about 15% decrease, about 1% decrease to about 10% decrease, about 1% decrease to about 5% decrease, about 5% decrease to about 99% decrease, an about 5% decrease to about 95% decrease, about 5% decrease to about 90% decrease, about 5% decrease to about 85% decrease, about 5% decrease to about 80% decrease, about 5% decrease to about 75% decrease, about 5% to about 70% decrease, about 5% decrease to about 65% decrease, about 5% decrease to about 60% decrease, about 5% decrease to about 55% decrease, about 5% decrease to about 50% decrease, about 5% decrease to about 45% decrease, about 5% decrease to about 40% decrease, about 5% decrease to about 35% decrease, about 5% decrease to about 30% decrease, about 5% decrease to about 25% decrease, about 5% decrease to about 20% decrease, about 5% decrease to about 15% decrease, about 5% decrease to about 10% decrease, about 10% decrease to about 99% decrease, an about 10% decrease to about 95% decrease, about 10% decrease to about 90% decrease, about 10% decrease to about 85% decrease, about 10% decrease to about 80% decrease, about 10% decrease to about 75% decrease, about 10% to about 70% decrease, about 10% decrease to about

65% decrease, about 10% decrease to about 60% decrease, about 10% decrease to about 55% decrease, about 10% decrease to about 50% decrease, about 10% decrease to about

45% decrease, about 10% decrease to about 40% decrease, about 10% decrease to about

35% decrease, about 10% decrease to about 30% decrease, about 10% decrease to about

25% decrease, about 10% decrease to about 20% decrease, about 10% decrease to about 15% decrease, about 15% decrease to about 99% decrease, an about 15% decrease to about 95% decrease, about 15% decrease to about 90% decrease, about 15% decrease to about 85% decrease, about 15% decrease to about 80% decrease, about 15% decrease to about 75% decrease, about 15% to about 70% decrease, about 15% decrease to about 65% decrease, about 15% decrease to about 60% decrease, about 15% decrease to about 55% decrease, about 15% decrease to about 50% decrease, about 15% decrease to about

45% decrease, about 15% decrease to about 40% decrease, about 15% decrease to about

35% decrease, about 15% decrease to about 30% decrease, about 15% decrease to about

25% decrease, about 15% decrease to about 20% decrease, about 20% decrease to about

99% decrease, an about 20% decrease to about 95% decrease, about 20% decrease to about 90% decrease, about 20% decrease to about 85% decrease, about 20% decrease to about 80% decrease, about 20% decrease to about 75% decrease, about 20% to about

70% decrease, about 20% decrease to about 65% decrease, about 20% decrease to about

60% decrease, about 20% decrease to about 55% decrease, about 20% decrease to about

50% decrease, about 20% decrease to about 45% decrease, about 20% decrease to about 40% decrease, about 20% decrease to about 35% decrease, about 20% decrease to about

30% decrease, about 20% decrease to about 25% decrease, about 25% decrease to about

99% decrease, an about 25% decrease to about 95% decrease, about 25% decrease to about 90% decrease, about 25% decrease to about 85% decrease, about 25% decrease to about 80% decrease, about 25% decrease to about 75% decrease, about 25% to about 70% decrease, about 25% decrease to about 65% decrease, about 25% decrease to about

60% decrease, about 25% decrease to about 55% decrease, about 25% decrease to about

50% decrease, about 25% decrease to about 45% decrease, about 25% decrease to about

40% decrease, about 25% decrease to about 35% decrease, about 25% decrease to about

30% decrease, about 30% decrease to about 99% decrease, an about 30% decrease to about 95% decrease, about 30% decrease to about 90% decrease, about 30% decrease to about 85% decrease, about 30% decrease to about 80% decrease, about 30% decrease to about 75% decrease, about 30% to about 70% decrease, about 30% decrease to about 65% decrease, about 30% decrease to about 60% decrease, about 30% decrease to about 55% decrease, about 30% decrease to about 50% decrease, about 30% decrease to about 45% decrease, about 30% decrease to about 40% decrease, about 30% decrease to about 35% decrease, about 35% decrease to about 99% decrease, an about 35% decrease to about 95% decrease, about 35% decrease to about 90% decrease, about 35% decrease to about 85% decrease, about 35% decrease to about 80% decrease, about 35% decrease to about 75% decrease, about 35% to about 70% decrease, about 35% decrease to about 65% decrease, about 35% decrease to about 60% decrease, about 35% decrease to about 55% decrease, about 35% decrease to about 50% decrease, about 35% decrease to about

45% decrease, about 35% decrease to about 40% decrease, about 40% decrease to about 99% decrease, an about 40% decrease to about 95% decrease, about 40% decrease to about 90% decrease, about 40% decrease to about 85% decrease, about 40% decrease to about 80% decrease, about 40% decrease to about 75% decrease, about 40% to about 70% decrease, about 40% decrease to about 65% decrease, about 40% decrease to about

60% decrease, about 40% decrease to about 55% decrease, about 40% decrease to about 50% decrease, about 40% decrease to about 45% decrease, about 45% decrease to about 99% decrease, an about 45% decrease to about 95% decrease, about 45% decrease to about 90% decrease, about 45% decrease to about 85% decrease, about 45% decrease to about 80% decrease, about 45% decrease to about 75% decrease, about 45% to about

70% decrease, about 45% decrease to about 65% decrease, about 45% decrease to about 60% decrease, about 45% decrease to about 55% decrease, about 45% decrease to about 50% decrease, about 50% decrease to about 99% decrease, an about 50% decrease to about 95% decrease, about 50% decrease to about 90% decrease, about 50% decrease to about 85% decrease, about 50% decrease to about 80% decrease, about 50% decrease to about 75% decrease, about 50% to about 70% decrease, about 50% decrease to about 65% decrease, about 50% decrease to about 60% decrease, about 50% decrease to about 55% decrease, about 55% decrease to about 99% decrease, an about 55% decrease to about 95% decrease, about 55% decrease to about 90% decrease, about 55% decrease to about 85% decrease, about 55% decrease to about 80% decrease, about 55% decrease to about 75% decrease, about 55% to about 70% decrease, about 55% decrease to about 65% decrease, about 55% decrease to about 60% decrease, about 60% decrease to about 99% decrease, an about 60% decrease to about 95% decrease, about 60% decrease to about 90% decrease, about 60% decrease to about 85% decrease, about 60% decrease to about 80% decrease, about 60% decrease to about 75% decrease, about 60% to about 70% decrease, about 60% decrease to about 65% decrease, about 65% decrease to about

99% decrease, an about 65% decrease to about 95% decrease, about 65% decrease to about 90% decrease, about 65% decrease to about 85% decrease, about 65% decrease to about 80% decrease, about 65% decrease to about 75% decrease, about 65% to about

70% decrease, about 70% decrease to about 99% decrease, an about 70% decrease to about 95% decrease, about 70% decrease to about 90% decrease, about 70% decrease to about 85% decrease, about 70% decrease to about 80% decrease, about 70% decrease to about 75% decrease, about 75% decrease to about 99% decrease, an about 75% decrease to about 95% decrease, about 75% decrease to about 90% decrease, about 75% decrease to about 85% decrease, about 75% decrease to about 80% decrease, about 80% decrease to about 99% decrease, an about 80% decrease to about 95% decrease, about 80% decrease to about 90% decrease, about 80% decrease to about 85% decrease, about 85% decrease to about 99% decrease, an about 85% decrease to about 95% decrease, about 85% decrease to about 90% decrease, about 90% decrease to about 99% decrease, an about 90% decrease to about 95% decrease, or about 95% decrease to about 99% decrease) in the number of senescent cells in the subject (e.g., a decrease in the number of senescent cells in one or more specific tissues involved and/or implicated in the aging- related disease or disorder in the subject), e.g., as compared to the number of senescent cells in the subject prior to treatment.

In some examples of these methods, the subject has been diagnosed or identified as having an infectious disease. Non-limiting examples of infectious disease include infection with human immunodeficiency virus, cytomegalovirus, adenovirus,

coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus. In some embodiments, these method can result in a decrease in the infectious titer (e.g., viral titer) in a subject (e.g., as compared to the infectious titer in the subject prior to treatment). In some embodiments, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of the infectious disease (e.g., viral infection) in the subject (e.g., as compared to the number, severity, or frequency of the one or more symptoms of the infectious disease in the subject prior to treatment).

The term“subject” refers to any mammal. In some embodiments, the subject or “subject in need of treatment” may be a canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), ovine, bovine, porcine, caprine, primate, e.g., a simian (e.g., a monkey (e.g., marmoset, baboon), or an ape (e.g., a gorilla, chimpanzee, orangutan, or gibbon) or a human; or rodent (e.g., a mouse, a guinea pig, a hamster, or a rat). In some

embodiments, the subject or“subject in need of treatment” may be a non-human mammal, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., murine, lapine, porcine, canine or primate animals) may be employed.

Methods of Killing a Cancer Cell, an Infected Cell, or a Senescent Cell

Also provided herein are methods of killing a cancer cell (e.g., any of the exemplary types of cancer described herein or known in the art), an infected cell (e.g., a cell infected with any of the exemplary viruses described herein or known in the art), or a senescent cell (e.g., a senescent cancer cell, a senescent fibroblast, or a senescent endothelial cell) in a subject in need thereof (e.g., any of the exemplary subjects described herein or known in the art) that include administering to the subject a therapeutically effective amount of any of the single-chain chimeric polypeptides or any of the multi-chain chimeric polypeptides described herein or any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, the subject can further be administered a therapeutically effective amount of any of the of IgGl antibody constructs described herein.

In some embodiments of these methods, the subject has been identified or diagnosed as having a cancer. Non-limiting examples of cancer include: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples of these methods, the subject has been identified or diagnosed as having an aging-related disease or condition. Non-limiting examples of aging-related diseases and conditions include Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples of these methods, the subject has been diagnosed or identified as having an infectious disease. Non-limiting examples of an infectious disease include infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Senescent Cells

Senescence is a form of irreversible growth arrest accompanied by phenotypic changes, resistance to apoptosis and activation of damage-sensing signaling pathways. Cellular senescence was first described in cultured human fibroblast cells that lost their ability to proliferate, reaching permanent arrest after about 50 population doublings (referred to as the Hayflick limit). Senescence is considered a stress response that can be induced by a wide range of intrinsic and extrinsic insults, including oxidative and genotoxic stress, DNA damage, telomere attrition, oncogenic activation, mitochondrial dysfunction, or chemotherapeutic agents.

Senescent cells remain metabolically active and can influence the tissue hemostasis, disease and aging through their secretory phenotype. Senescence is considered as a physiologic process and is important in promoting wound healing, tissue homeostasis, regeneration, and fibrosis regulation. For instance, transient induction of senescent cells is observed during would healing and contributes to wound resolution. Perhaps one of the most important roles of senescence is its role in tumor suppression. However, the accumulation of senescent cells also drives aging and aging-related diseases and conditions. The senescent phenotype also can trigger chronic inflammatory responses and consequently augment chronic inflammatory conditions to promote tumor growth. The connection between senescence and aging was initially based on

observations that senescent cells accumulate in aged tissue. The use of transgenic models has enabled the detection of senescent cells systematically in many age-related pathologies. Strategies to selectively eliminate senescent cells have demonstrated that senescent cells can indeed play a causal role in aging and related pathologies.

Senescent cells display important and unique properties which include changes in morphology, chromatin organization, gene expression, and metabolism. There are several biochemical and functional properties associated with cellular senescence, such as (i) increased expression of pl6 and p2l, inhibitors of cyclin-dependent kinases, (ii) presence of senescence-associated b-galactosidase, a marker of lysosomal activity, (iii) appearance of senescence-associated heterochromatin foci and downregulation of lamin Bl levels, (iv) resistance to apoptosis caused by an increased expression of anti-apoptotic BCL-family protein, and (v) upregulation of CD26 (DPP4), CD36 (Scavenger receptor), forkhead box 4 (FOX04), and secretory carrier membrane protein 4 (SCAMP4).

Senescent cells also express an inflammatory signature, the so-called senescence- associated secretory phenotype (SASP). Through SASP, the senescent cells produce a wide range of inflammatory cytokines (IL-6, IL-8), growth factors (TGF-b), chemokines (CCL-2), and matrix metalloproteinases (MMP-3, MMP-9) that operate in a cell- autonomous manner to reinforce senescence (autocrine effects) and communicate with and modify the microenvironment (paracrine effects). SASP factors can contribute to tumor suppression by triggering senescence surveillance, an immune-mediated clearance of senescent cells. However, chronic inflammation is also a known driver of

tumorigenesis, and accumulating evidence indicates that chronic SASP can also boost cancer and aging-related diseases.

The secretion profile of senescent cells is context dependent. For instance, the mitochondrial dysfunction-associated senescence (MiDAS), induced by different mitochondrial dysfunction in human fibroblasts, led to the appearance of a SASP that was deficient in IL-l -dependent inflammatory factors. A decrease in the NAD+/NADH ratio activated AMPK signaling which induced MiDAS through the activation of p53. As a result, p53 inhibited NF-kB signaling which is a crucial inducer of pro-inflammatory SASP. In contrast, the cellular senescence caused by persistent DNA damage in human cells induced an inflammatory SASP, which was dependent on the activation of ataxia- telangiectasia mutated (ATM) kinase but not on that of p53. In particular, the expression and secretion levels of IL-6 and IL-8 were increased. It was also demonstrated that cellular senescence caused by the ectopic expression pl6INK4a and p2lCIPl induced the senescent phenotype in human fibroblasts without an inflammatory SASP indicating that the growth arrest itself did not stimulate SASP.

One of the most defining characteristics of senescence is stable growth arrest.

This is achieved by two important pathways, the pl6/Rb and the p53/p2l, both of which are central in tumor suppression. DNA damage results in: (1) high deposition of gH2Ac (histone coding gene) and 53BP1 (involved in DNA damage response) in chromatin: this leads to activation of a kinase cascade eventually resulting in p53 activation, and (2) activation of pl6INK4a and ARF (both encoded by CDKN2A) and Pl5INK4b (encoded by CDKN2B): p53 induces transcription of cyclin-dependent kinase inhibitor (p2l) and along with both pl6INK4a and pl5INK4b block genes for cell cycle progression (CDK4 and CDK6). This eventually leads to hypophosphorylation of Retinoblastoma protein (Rb) and cell cycle arrest at the Gl phase.

Selectively killing senescent cells has been shown to significantly improve the health span of mice in the context of normal aging and ameliorates the consequences of age-related disease or cancer therapy (Ovadya, J Clin Invest. 128(4): 1247-1254, 2018). In nature, the senescent cells are normally removed by the innate immune cells. Induction of senescence not only prevents the potential proliferation and transformation of damaged/altered cells, but also favors tissue repair through the production of SASP factors that function as chemoattractants mainly for Natural Killer (NK) cells (such as IL- 15 and CCL2) and macrophages (such as CFS-l and CCL2). These innate immune cells mediate the immunosurveillance mechanism for eliminating stressed cells. Senescent cells usually up-regulate the NK-cell activating receptor NKG2D and DNAM-l ligands, which belong to a family of stress-inducible ligands: an important component of the frontline immune defense against infectious diseases and malignancies. Upon receptor activation, NK cells can then specifically induce the death of senescent cells through their cytolytic machinery. A role for NK cells in the immune surveillance of senescent cells has been pointed out in liver fibrosis (Sagiv, Oncogene 32(15): 1971-1977, 2013), hepatocellular carcinoma (Iannello, J Exp Med 210(10): 2057-2069, 2013), multiple myeloma (Soriani, Blood 113(15): 3503-3511, 2009), and glioma cells stressed by dysfunction of the mevalonate pathway (Ciaglia, Int J Cancer 142(1): 176-190, 2018). Endometrial cells undergo acute cellular senescence and do not differentiate into decidual cells. The differentiated decidual cells secrete IL-15 and thereby recruit uterine NK cells to target and eliminate the undifferentiated senescent cells thus helping to re-model and rejuvenate the endometrium (Brighton, Elife 6: e3 l274, 2017). With a similar mechanism, during liver fibrosis, p53-expressing senescent liver satellite cells skewed the polarization of resident Kupfer macrophages and freshly infiltrated macrophages toward the pro-inflammatory Ml phenotype, which display senolytic activity. F4/80+ macrophages have been shown to play a key role in the clearance of mouse uterine senescent cells to maintain postpartum uterine function.

Senescent cells recruit NK cells by mainly upregulating ligands to NKG2D (expressed on NK cells), chemokines, and other SASP factors. In vivo models of liver fibrosis have shown effective clearance of senescent cells by activated NK cells

(Krizhanovsky, Cell 134(4): 657-667, 2008). Studies have described various models to study senescence including liver fibrosis (Krizhanovsky, Cell 134(4): 657-667, 2008), osteoarthritis (Xu, J Gerontol A Biol SciMed Sci 72(6): 780-785, 2017), and Parkinson’s disease (Chinta, Cell Rep 22(4): 930-940, 2018). Animal models for studying senescent cells are described in: Krizhanovsky, Cell 134(4): 657-667, 2008; Baker, Nature

All 479(7372): 232-236, 2011; Farr, Nat Med 23(9): 1072-1079, 2017; Bourgeois, FEES Lett 592(12): 2083-2097, 2018; Xu, NatMed24(%): 1246-1256, 2018).

Methods of Increasing Glucose Consumption of an Immune Cell

Also provided herein are methods of increasing the glucose consumption of an immune cell that include: contacting an immune cell (e.g., any of the immune cells described herein) in a liquid culture medium (e.g., any of the liquid culture media described herein) including an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain (e.g., any of the single-chain chimeric polypeptides described herein), and optionally (ii) an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to the linker domain (e.g.., any of the exemplary IgGl antibody constructs described herein), under conditions that allow for glucose

consumption in the immune cell, where the first target-binding domain and the second target-binding domain are each independently selected from the group of: a soluble interleukin or cytokine protein, an antigen-binding domain, a soluble interleukin or cytokine receptor, and ligands of co-stimulatory molecules.

Also provided herein are methods of increasing the glucose consumption of an immune cell that include: contacting an immune cell in a liquid culture medium comprising (1) an effective amount of a multi-chain chimeric polypeptide including: (a) a first chimeric polypeptide including: (i) a first target-binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, where the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains (e.g., any of the multi-chain chimeric polypeptides described herein); and (2) an effective amount of an IgGl antibody construct including at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for glucose consumption in the immune cell.

In some embodiments of any of the methods described herein, the increase in glucose consumption is compared to the level of glucose consumption in a similar immune cell not contacted with the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide, and optionally the IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to the soluble tissue factor domain. Non-limiting assays that can be used to detect glucose consumption include, e.g., assays that include the use of radiolabeled 3-O-methylglucose or radiolabeled 2-deoxy-glucose, or an enzymatic, fluorometric assay for detecting 2-deoxyglucose. Additional examples of assays that can be used to detect glucose consumption are described in, e.g.,

Yamamoto et al., Curr. Protoc. Pharmacol ., Chapter 12, Unit 12.14.1-22, December 2011; Zou et al., J Biochem. Biophys. Methods 64(3):207-2l5, September 2005; and MacKrell et al., Diabetes p. 1-9, published online on March 13, 2012.

In some embodiments of these methods, the liquid culture medium includes the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide, and the IgGl antibody construct. In some examples of these methods, the IgGl antibody construct is a monoclonal IgGl antibody, where both of the antigen-binding domains in the monoclonal IgGl antibody bind specifically to the soluble tissue factor domain. In some examples of these methods, the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the soluble tissue factor domain.

In some embodiments of any of the methods described herein, the contacting step is performed for a period of about 2 hours to about 20 days (e.g., about 2 hours to about 18 days, about 2 hours to about 16 days, about 2 hours to about 14 days, about 2 hours to about 12 days, about 2 hours to about 10 days, about 2 hours to about 8 days, about 2 hours to about 7 days, about 2 hours to about 6 days, about 2 hours to about 5 days, about 2 hours to about 4 days, about 2 hours to about 3 days, about 2 hours to about 2 days, about 2 hours to about 1 day, about 6 hours to about 18 days, about 6 hours to about 16 days, about 6 hours to about 14 days, about 6 hours to about 12 days, about 6 hours to about 10 days, about 6 hours to about 8 days, about 6 hours to about 7 days, about 6 hours to about 6 days, about 6 hours to about 5 days, about 6 hours to about 4 days, about 6 hours to about 3 days, about 6 hours to about 2 days, about 6 hours to about 1 day, about 12 hours to about 18 days, about 12 hours to about 16 days, about 12 hours to about 14 days, about 12 hours to about 12 days, about 12 hours to about 10 days, about 12 hours to about 8 days, about 12 hours to about 7 days, about 12 hours to about 6 days, about 12 hours to about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12 hours to about 2 days, about 12 hours to about 1 day, about 1 day to about 18 days, about 1 day to about 16 days, about 1 day to about 15 days, about 1 day to about 14 days, about 1 day to about 12 days, about 1 day to about 10 days, about 1 day to about

8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 18 days, about 2 days to about 16 days, about 2 days to about 14 days, about 2 days to about 12 days, about 2 days to about 10 days, about 2 days to about 8 days, about 2 days to about 7 days, about 2 days to about 6 days, about 2 days to about 5 days, about 2 days to about 4 days, about 2 days to about 3 days, about 3 days to about 18 days, about 3 days to about 16 days, about 3 days to about 14 days, about 3 days to about 12 days, about 3 days to about 10 days, about 3 days to about 8 days, about 3 days to about 7 days, about 3 days to about 6 days, about 3 days to about 5 days, about 3 days to about 4 days, about 4 days to about 18 days, about 4 days to about 16 days, about 4 days to about 14 days, about 4 days to about 12 days, about 4 days to about 10 days, about 4 days to about 8 days, about 4 days to about 7 days, about 4 days to about 6 days, about 4 days to about 5 days, about 5 days to about 18 days, about 5 days to about 16 days, about 5 days to about 14 days, about 5 days to about 12 days, about 5 days to about 10 days, about 5 days to about 8 days, about 5 days to about 7 days, about 5 days to about

6 days, about 6 days to about 18 days, about 6 days to about 16 days, about 6 days to about 14 days, about 6 days to about 12 days, about 6 days to about 10 days, about 6 days to about 8 days, about 6 days to about 7 days, about 7 days to about 18 days, about 7 days to about 16 days, about 7 days to about 14 days, about 7 days to about 12 days, about 7 days to about 10 days, about 7 days to about 8 days, about 8 days to about 18 days, about 8 days to about 16 days, about 8 days to about 14 days, about 8 days to about 12 days, about 8 days to about 10 days, about 9 days to about 18 days, about 9 days to about 16 days, about 9 days to about 14 days, about 9 days to about 12 days, about 12 days to about 18 days, about 12 days to about 16 days, about 12 days to about 14 days, about 14 days to about 18 days, about 14 days to about 16 days, or about 16 days to about 18 days). In some embodiments of these methods, the liquid culture medium can be a serum-free liquid culture medium. In some embodiments of these methods, the liquid culture medium can be a chemically-defined liquid culture medium. In other examples of these methods, the liquid culture medium includes serum.

In some examples of these methods, the liquid culture medium includes the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide, and the IgGl antibody construct at a molar ratio of about 0.5: 1 to about 2: 1 (e.g., about 0.8: 1 to about 1.2: 1).

In some embodiments of these methods, the immune cell is selected from the group of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Thl7 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, gd T cell, an ab T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell. In some embodiments of any of the methods described herein, the immune cell is a T-cell or a natural killer cell.

In some examples of these methods, the immune cell was previously obtained from a subject. Some embodiments of these methods further include obtaining the immune cell from the subject prior to the contacting step. In some embodiments of these methods, the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor. Some embodiments of these methods further include, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Some

embodiments of these methods further include, before the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a

recombinant T-cell receptor. Some embodiments of these methods further include, after the contacting step, isolating the immune cell. Some embodiments of these methods further include, after the contacting step, administering the immune cell to a subject in need thereof.

In some examples of these methods, the subject has been identified or diagnosed as having an age-related disease or condition. In some examples of these methods, the age-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some examples of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples of these methods, the subject has been diagnosed or identified as having an infectious disease. In some examples of these methods, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, hepatitis C virus, papillomavirus, or influenza virus.

Also provided herein are activated immune cells produced by any of these methods. Also provided herein are pharmaceutical compositions that include any of the activated immune cells produced by any of these methods. Also provided herein are kits that include any of the pharmaceutical compositions described herein produced by any of the methods described herein.

Also provided herein are methods of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced by any of the methods described herein or any of the pharmaceutical compositions described herein.

In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some examples of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples of these methods, the subject has been identified or diagnosed as having an aging-related disease or condition. In some examples of these methods, the aging-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction Also provided herein are methods of treating a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced by any of the methods described herein or any of the pharmaceutical compositions described herein. In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some embodiments of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,

rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some embodiments of these methods, the subject has been identified or diagnosed as having an aging-related disease or condition. In some examples of these methods, the aging-related disease or condition is selected from the group of:

Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis,

Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples of these methods, the subject has been diagnosed or identified as having an infectious disease. In some examples of these methods, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, hepatitis C virus, papillomavirus, or influenza virus. Methods of Increasing Oxidative Phosphorylation of an Immune Cell

Also provided herein are methods of increasing the oxidative phosphorylation of an immune cell that include: contacting an immune cell (e.g., any of the immune cells described herein) in a liquid culture medium (e.g., any of the liquid culture media described herein) including an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain (e.g., any of the exemplary single-chain chimeric polypeptides described herein), and optionally (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain (e.g.., any of the exemplary IgGl antibody constructs described herein), under conditions that allow for oxidative phosphorylation in the immune cell, where the first target-binding domain and the second target-binding domain are each independently selected from the group of: a soluble interleukin or cytokine protein, an antigen-binding domain, a soluble interleukin or cytokine receptor, and ligands of co- stimulatory molecules.

Also provided herein are methods of increasing the oxidative phosphorylation of an immune cell that include: contacting an immune cell (e.g., any of the immune cells described herein) in a liquid culture medium (e.g., any of the liquid culture media described herein) comprising (1) an effective amount of a multi-chain chimeric polypeptide including: (a) a first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, where the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains (e.g., any of the multi-chain chimeric polypeptides described herein); and (2) an effective amount of an IgGl antibody construct including at least one antigen-binding domain that binds specifically to the linker domain (e.g.., any of the exemplary IgGl antibody constructs described herein), under conditions that allow for oxidative phosphorylation in the immune cell.

In some embodiments of any of the methods described herein, the increase in oxidative phosphorylation is compared to the level of oxidative phosphorylation in a similar immune cell not contacted with the single-chain chimeric polypeptide of the multi-chain chimeric polypeptide, and optionally the IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to the soluble tissue factor domain.

Non-limiting assays that can be used to detect oxidative phosphorylation include, e.g., immunofluorescent assays and colorimetric assays. A non-limiting commercial assay that can be used to determine the level of oxidative phosphorylation is MitoTox™ Complete OXPHOS Activity Assay (Abeam). Additional examples of assays that can be used to detect oxidative phosphorylation are described in, e.g., Chance et al., Nature 175: 1120-1121, 1955; Pullman et al., Science 123(3208): 1105-1107, 1956; Van Bergen et al., Mitochondrion 15:24-33, 2014; and Rocha et al., Sci. Rep. 5: 15037, 2015.

In some embodiments of these methods, the liquid culture medium includes the single chain chimeric polypeptide or the multi-chain chimeric polypeptide, and the IgGl antibody construct. In some examples of these methods, the IgGl antibody construct is a monoclonal IgGl antibody, where both of the antigen-binding domains in the monoclonal IgGl antibody bind specifically to the soluble tissue factor domain. In some examples of these methods, the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the soluble tissue factor domain.

In some embodiments of any of the methods described herein, the contacting step is performed for a period of about 2 hours to about 20 days (e.g., about 2 hours to about 18 days, about 2 hours to about 16 days, about 2 hours to about 14 days, about 2 hours to about 12 days, about 2 hours to about 10 days, about 2 hours to about 8 days, about 2 hours to about 7 days, about 2 hours to about 6 days, about 2 hours to about 5 days, about 2 hours to about 4 days, about 2 hours to about 3 days, about 2 hours to about 2 days, about 2 hours to about 1 day, about 6 hours to about 18 days, about 6 hours to about 16 days, about 6 hours to about 14 days, about 6 hours to about 12 days, about 6 hours to about 10 days, about 6 hours to about 8 days, about 6 hours to about 7 days, about 6 hours to about 6 days, about 6 hours to about 5 days, about 6 hours to about 4 days, about 6 hours to about 3 days, about 6 hours to about 2 days, about 6 hours to about 1 day, about 12 hours to about 18 days, about 12 hours to about 16 days, about 12 hours to about 14 days, about 12 hours to about 12 days, about 12 hours to about 10 days, about 12 hours to about 8 days, about 12 hours to about 7 days, about 12 hours to about 6 days, about 12 hours to about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12 hours to about 2 days, about 12 hours to about 1 day, about 1 day to about 18 days, about 1 day to about 16 days, about 1 day to about 15 days, about 1 day to about 14 days, about 1 day to about 12 days, about 1 day to about 10 days, about 1 day to about

8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 18 days, about 2 days to about 16 days, about 2 days to about 14 days, about 2 days to about 12 days, about 2 days to about 10 days, about 2 days to about 8 days, about 2 days to about 7 days, about 2 days to about 6 days, about 2 days to about 5 days, about 2 days to about 4 days, about 2 days to about 3 days, about 3 days to about 18 days, about 3 days to about 16 days, about 3 days to about 14 days, about 3 days to about 12 days, about 3 days to about 10 days, about 3 days to about 8 days, about 3 days to about 7 days, about 3 days to about 6 days, about 3 days to about 5 days, about 3 days to about 4 days, about 4 days to about 18 days, about 4 days to about 16 days, about 4 days to about 14 days, about 4 days to about 12 days, about 4 days to about 10 days, about 4 days to about 8 days, about 4 days to about 7 days, about 4 days to about 6 days, about 4 days to about 5 days, about 5 days to about 18 days, about 5 days to about 16 days, about 5 days to about 14 days, about 5 days to about 12 days, about 5 days to about 10 days, about 5 days to about 8 days, about 5 days to about 7 days, about 5 days to about

6 days, about 6 days to about 18 days, about 6 days to about 16 days, about 6 days to about 14 days, about 6 days to about 12 days, about 6 days to about 10 days, about 6 days to about 8 days, about 6 days to about 7 days, about 7 days to about 18 days, about 7 days to about 16 days, about 7 days to about 14 days, about 7 days to about 12 days, about 7 days to about 10 days, about 7 days to about 8 days, about 8 days to about 18 days, about 8 days to about 16 days, about 8 days to about 14 days, about 8 days to about 12 days, about 8 days to about 10 days, about 9 days to about 18 days, about 9 days to about 16 days, about 9 days to about 14 days, about 9 days to about 12 days, about 12 days to about 18 days, about 12 days to about 16 days, about 12 days to about 14 days, about 14 days to about 18 days, about 14 days to about 16 days, or about 16 days to about 18 days).

In some embodiments of these methods, the liquid culture medium can be a serum-free liquid culture medium. In some embodiments of these methods, the liquid culture medium can be a chemically-defined liquid culture medium. In other examples of these methods, the liquid culture medium includes serum.

In some examples of these methods, the liquid culture medium includes the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide, and the IgGl antibody construct at a molar ratio of about 0.5:1 to about 2: 1 (e.g., about 0.8: 1 to about 1.2: 1).

In some embodiments of these methods, the immune cell is selected from the group of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Thl7 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, gd T cell, an ab T cell, a tumor- infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell. In some embodiments of any of the methods described herein, the immune cell is a T-cell or a natural killer cell.

In some examples of these methods, the immune cell was previously obtained from a subject. Some embodiments of these methods further include obtaining the immune cell from the subject prior to the contacting step. In some embodiments of these methods, the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor. Some embodiments of these methods further include, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Some

embodiments of these methods further include, before the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a

recombinant T-cell receptor. Some embodiments of these methods further include, after the contacting step, isolating the immune cell. Some embodiments of these methods further include, after the contacting step, administering the immune cell to a subject in need thereof.

In some examples of these methods, the subject has been identified or diagnosed as having an age-related disease or condition. In some examples of these methods, the age-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some examples of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples of these methods, the subject has been diagnosed or identified as having an infectious disease. In some examples of these methods, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, hepatitis C virus, papillomavirus, or influenza virus. Also provided herein are activated immune cells produced by any of these methods. Also provided herein are pharmaceutical compositions that include any of the activated immune cells produced by any of these methods. Also provided herein are kits that include any of the pharmaceutical compositions described herein produced by any of the methods described herein.

Also provided herein are methods of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced by any of the methods described herein or any of the pharmaceutical compositions described herein.

In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some examples of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples of these methods, the subject has been identified or diagnosed as having an aging-related disease or condition. In some examples of these methods, the aging-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction

Also provided herein are methods of treating a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced by any of the methods described herein or any of the pharmaceutical compositions described herein. In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some embodiments of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,

rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some embodiments of these methods, the subject has been identified or diagnosed as having an aging-related disease or condition. In some examples of these methods, the aging-related disease or condition is selected from the group of:

Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis,

Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples of these methods, the subject has been diagnosed or identified as having an infectious disease. In some examples of these methods, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, hepatitis C virus, papillomavirus, or influenza virus.

Methods of Increasing Aerobic Glycolysis of an Immune Cell

Also provided herein are methods of increasing the aerobic glycolysis of an immune cell that include: contacting an immune cell (e.g., any of the immune cells described herein) in a liquid culture medium (e.g., any of the liquid culture media described herein) including an effective amount of (i) a single-chain chimeric polypeptide including a first target-binding domain, a linker domain, and a second target-binding domain (e.g., any of the single-chain chimeric polypeptides described herein), and optionally (ii) an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to the linker domain (e.g., any of the exemplary IgGl antibody constructs described herein), under conditions that allow for aerobic glycolysis in the immune cell, where the first target-binding domain and the second target-binding domain are each independently selected from the group of: a soluble interleukin or cytokine protein, an antigen-binding domain, a soluble interleukin or cytokine receptor, and ligands of co-stimulatory molecules.

Also provided herein are methods of increasing the aerobic glycolysis of an immune cell that include: contacting an immune cell (e.g., any of the immune cells described herein) in a liquid culture medium (e.g., any of the liquid culture media described herein) including (1) an effective amount of a multi-chain chimeric polypeptide including: (a) a first chimeric polypeptide including: (i) a first target-binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, where the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains (e.g., any of the multi-chain chimeric polypeptides described herein); and (2) an effective amount of an IgGl antibody construct including at least one antigen-binding domain that binds specifically to the linker domain (e.g.., any of the exemplary IgGl antibody constructs described herein), under conditions that allow for aerobic glycolysis in the immune cell.

In some embodiments of any of the methods described herein, the increase in aerobic glycolysis is compared to the level of aerobic glycolysis in a similar immune cell not contacted with the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide, and optionally the IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to the soluble tissue factor domain.

Non-limiting assays that can be used to detect aerobic glycolysis include, e.g., detection of ATP and/or CO2 production, e.g., using gas chromatography/mass spectrometry, or extracellular acidification rate. Additional examples of assays that can be used to detect aerobic glycolysis are described in, e.g., Zhang et al., Bone 114:150- 160, 2018; Zhao et al., J Breast Cancer 21(2): 112-123, 2018; Li et al., Cell Comm.

Signal. l6(l):26, 2018; Cai et al., J. Exp. Clin. Cancer Res. 37(1): 104, 2018; and Zhang et al., Oncol. Rep. 40(2): 1156-1164, 2018.

In some embodiments of these methods, the liquid culture medium includes the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide, and the IgGl antibody construct. In some examples of these methods, the IgGl antibody construct is a monoclonal IgGl antibody, where both of the antigen-binding domains in the monoclonal IgGl antibody bind specifically to the soluble tissue factor domain. In some examples of these methods, the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the soluble tissue factor domain.

In some embodiments of any of the methods described herein, the contacting step is performed for a period of about 2 hours to about 20 days (e.g., about 2 hours to about 18 days, about 2 hours to about 16 days, about 2 hours to about 14 days, about 2 hours to about 12 days, about 2 hours to about 10 days, about 2 hours to about 8 days, about 2 hours to about 7 days, about 2 hours to about 6 days, about 2 hours to about 5 days, about 2 hours to about 4 days, about 2 hours to about 3 days, about 2 hours to about 2 days, about 2 hours to about 1 day, about 6 hours to about 18 days, about 6 hours to about 16 days, about 6 hours to about 14 days, about 6 hours to about 12 days, about 6 hours to about 10 days, about 6 hours to about 8 days, about 6 hours to about 7 days, about 6 hours to about 6 days, about 6 hours to about 5 days, about 6 hours to about 4 days, about 6 hours to about 3 days, about 6 hours to about 2 days, about 6 hours to about 1 day, about 12 hours to about 18 days, about 12 hours to about 16 days, about 12 hours to about 14 days, about 12 hours to about 12 days, about 12 hours to about 10 days, about 12 hours to about 8 days, about 12 hours to about 7 days, about 12 hours to about 6 days, about 12 hours to about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12 hours to about 2 days, about 12 hours to about 1 day, about 1 day to about 18 days, about 1 day to about 16 days, about 1 day to about 15 days, about 1 day to about 14 days, about 1 day to about 12 days, about 1 day to about 10 days, about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 18 days, about 2 days to about 16 days, about 2 days to about

14 days, about 2 days to about 12 days, about 2 days to about 10 days, about 2 days to about 8 days, about 2 days to about 7 days, about 2 days to about 6 days, about 2 days to about 5 days, about 2 days to about 4 days, about 2 days to about 3 days, about 3 days to about 18 days, about 3 days to about 16 days, about 3 days to about 14 days, about 3 days to about 12 days, about 3 days to about 10 days, about 3 days to about 8 days, about 3 days to about 7 days, about 3 days to about 6 days, about 3 days to about 5 days, about 3 days to about 4 days, about 4 days to about 18 days, about 4 days to about 16 days, about 4 days to about 14 days, about 4 days to about 12 days, about 4 days to about 10 days, about 4 days to about 8 days, about 4 days to about 7 days, about 4 days to about 6 days, about 4 days to about 5 days, about 5 days to about 18 days, about 5 days to about 16 days, about 5 days to about 14 days, about 5 days to about 12 days, about 5 days to about 10 days, about 5 days to about 8 days, about 5 days to about 7 days, about 5 days to about 6 days, about 6 days to about 18 days, about 6 days to about 16 days, about 6 days to about 14 days, about 6 days to about 12 days, about 6 days to about 10 days, about 6 days to about 8 days, about 6 days to about 7 days, about 7 days to about 18 days, about 7 days to about 16 days, about 7 days to about 14 days, about 7 days to about 12 days, about 7 days to about 10 days, about 7 days to about 8 days, about 8 days to about 18 days, about 8 days to about 16 days, about 8 days to about 14 days, about 8 days to about 12 days, about 8 days to about 10 days, about 9 days to about 18 days, about 9 days to about 16 days, about 9 days to about 14 days, about 9 days to about 12 days, about 12 days to about 18 days, about 12 days to about 16 days, about 12 days to about 14 days, about 14 days to about 18 days, about 14 days to about 16 days, or about 16 days to about 18 days).

In some embodiments of these methods, the liquid culture medium can be a serum-free liquid culture medium. In some embodiments of these methods, the liquid culture medium can be a chemically-defined liquid culture medium. In other examples of these methods, the liquid culture medium includes serum.

In some examples of these methods, the liquid culture medium includes the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide, and the IgGl antibody construct at a molar ratio of about 0.5: 1 to about 2: 1 (e.g., about 0.8: 1 to about 1.2: 1).

In some embodiments of these methods, the immune cell is selected from the group of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Thl7 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, gd T cell, an ab T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell. In some embodiments of any of the methods described herein, the immune cell is a T-cell or a natural killer cell.

In some examples of these methods, the immune cell was previously obtained from a subject. Some embodiments of these methods further include obtaining the immune cell from the subject prior to the contacting step. In some embodiments of these methods, the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor. Some embodiments of these methods further include, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Some

embodiments of these methods further include, before the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a

recombinant T-cell receptor. Some embodiments of these methods further include, after the contacting step, isolating the immune cell. Some embodiments of these methods further include, after the contacting step, administering the immune cell to a subject in need thereof.

In some examples of these methods, the subject has been identified or diagnosed as having an age-related disease or condition. In some examples of these methods, the age-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some examples of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples of these methods, the subject has been diagnosed or identified as having an infectious disease. In some examples of these methods, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, hepatitis C virus, papillomavirus, or influenza virus.

Also provided herein are activated immune cells produced by any of these methods. Also provided herein are pharmaceutical compositions that include any of the activated immune cells produced by any of these methods. Also provided herein are kits that include any of the pharmaceutical compositions described herein produced by any of the methods described herein.

Also provided herein are methods of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced by any of the methods described herein or any of the pharmaceutical compositions described herein.

In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some examples of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma,

endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples of these methods, the subject has been identified or diagnosed as having an aging-related disease or condition. In some examples of these methods, the aging-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction

Also provided herein are methods of treating a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced by any of the methods described herein or any of the pharmaceutical compositions described herein. In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some embodiments of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,

rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some embodiments of these methods, the subject has been identified or diagnosed as having an aging-related disease or condition. In some examples of these methods, the aging-related disease or condition is selected from the group of:

Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples of these methods, the subject has been diagnosed or identified as having an infectious disease. In some examples of these methods, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, hepatitis C virus, papillomavirus, or influenza virus.

Methods of Increasing Extracellular Acidification Rate (ECAR) of an Immune Cell Also provided herein are method of increasing the extracellular acidification rate (ECAR) of an immune cell that include: contacting an immune cell (e.g., any of the immune cells described herein) in a liquid culture medium (e.g., any of the liquid culture media described herein) including an effective amount of (i) a single-chain chimeric polypeptide including a first target-binding domain, a linker domain, and a second target- binding domain (e.g., any of the single-chain chimeric polypeptides described herein), and optionally (ii) an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to the linker domain (e.g., any of the exemplary IgGl antibody constructs described herein), under conditions that allow for extracellular acidification by the immune cell, where the first target-binding domain and the second target-binding domain are each independently selected from the group of: a soluble interleukin or cytokine protein, an antigen-binding domain, a soluble interleukin or cytokine receptor, and ligands of co-stimulatory molecules.

Also provided herein are methods of increasing the extracellular acidification rate

(ECAR) of an immune cell that include: contacting an immune cell (e.g., any of the immune cells described herein) in a liquid culture medium (e.g., any of the liquid culture media described herein) including (1) an effective amount of a multi-chain chimeric polypeptide including: (a) a first chimeric polypeptide including: (i) a first target-binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, where the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains (e.g., any of the multi-chain chimeric polypeptides described herein); and (2) an effective amount of an IgGl antibody construct including at least one antigen-binding domain that binds specifically to the linker domain (e.g., any of the exemplary IgGl antibody constructs described herein), under conditions that allow for extracellular acidification by the immune cell.

In some embodiments of any of the methods described herein, the increase in extracellular acidification rate (ECAR) is compared to the level of extracellular acidification rate (ECAR) in a similar immune cell not contacted with the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide, and optionally the IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to the soluble tissue factor domain.

Non-limiting commercial assays that can be used to detect extracellular acidification rate (ECAR) include, e.g., Glycolysis Assay (Extracellular Acidification Kit) (ab 197244 or ab 197245) (Abeam), MITO-ID® Extracellular pH Sensor Kit (ENZ- 51048) (Enzo Life Sciences), and pH-Xtra™- Glycolysis Assay (Extracellular

Acidification). Additional examples of assays that can be used to detect extracellular acidification rate (ECAR) are described in, e.g., Owicki et ah, Biosens. Bioelectron. 7(4):255-272, 1992; Hynes et ah, Analytical Biochem. 390(l):2l-28, 2009; Trevani et ah,

J. Immunol. l62(8):4849-4857, 1999; and Mookerjee et ah, J. Vis. Exp. 106:53464, 2015.

In some embodiments of these methods, the liquid culture medium includes the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide, and the IgGl antibody construct. In some examples of these methods, the IgGl antibody construct is a monoclonal IgGl antibody, where both of the antigen-binding domains in the monoclonal IgGl antibody bind specifically to the soluble tissue factor domain. In some examples of these methods, the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the soluble tissue factor domain.

In some embodiments of any of the methods described herein, the contacting step is performed for a period of about 2 hours to about 20 days (e.g., about 2 hours to about 18 days, about 2 hours to about 16 days, about 2 hours to about 14 days, about 2 hours to about 12 days, about 2 hours to about 10 days, about 2 hours to about 8 days, about 2 hours to about 7 days, about 2 hours to about 6 days, about 2 hours to about 5 days, about 2 hours to about 4 days, about 2 hours to about 3 days, about 2 hours to about 2 days, about 2 hours to about 1 day, about 6 hours to about 18 days, about 6 hours to about 16 days, about 6 hours to about 14 days, about 6 hours to about 12 days, about 6 hours to about 10 days, about 6 hours to about 8 days, about 6 hours to about 7 days, about 6 hours to about 6 days, about 6 hours to about 5 days, about 6 hours to about 4 days, about 6 hours to about 3 days, about 6 hours to about 2 days, about 6 hours to about 1 day, about 12 hours to about 18 days, about 12 hours to about 16 days, about 12 hours to about 14 days, about 12 hours to about 12 days, about 12 hours to about 10 days, about 12 hours to about 8 days, about 12 hours to about 7 days, about 12 hours to about 6 days, about 12 hours to about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12 hours to about 2 days, about 12 hours to about 1 day, about 1 day to about 18 days, about 1 day to about 16 days, about 1 day to about 15 days, about 1 day to about

14 days, about 1 day to about 12 days, about 1 day to about 10 days, about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 18 days, about 2 days to about 16 days, about 2 days to about 14 days, about 2 days to about 12 days, about 2 days to about 10 days, about 2 days to about 8 days, about 2 days to about 7 days, about 2 days to about 6 days, about 2 days to about 5 days, about 2 days to about 4 days, about 2 days to about 3 days, about 3 days to about 18 days, about 3 days to about 16 days, about 3 days to about 14 days, about 3 days to about 12 days, about 3 days to about 10 days, about 3 days to about 8 days, about 3 days to about 7 days, about 3 days to about 6 days, about 3 days to about 5 days, about 3 days to about 4 days, about 4 days to about 18 days, about 4 days to about 16 days, about 4 days to about 14 days, about 4 days to about 12 days, about 4 days to about 10 days, about 4 days to about 8 days, about 4 days to about 7 days, about 4 days to about 6 days, about 4 days to about 5 days, about 5 days to about 18 days, about 5 days to about 16 days, about 5 days to about 14 days, about 5 days to about 12 days, about 5 days to about 10 days, about 5 days to about 8 days, about 5 days to about 7 days, about 5 days to about 6 days, about 6 days to about 18 days, about 6 days to about 16 days, about 6 days to about 14 days, about 6 days to about 12 days, about 6 days to about 10 days, about 6 days to about 8 days, about 6 days to about 7 days, about 7 days to about 18 days, about 7 days to about 16 days, about 7 days to about 14 days, about 7 days to about 12 days, about 7 days to about 10 days, about 7 days to about 8 days, about 8 days to about 18 days, about 8 days to about 16 days, about 8 days to about 14 days, about 8 days to about 12 days, about 8 days to about 10 days, about 9 days to about 18 days, about 9 days to about 16 days, about 9 days to about 14 days, about 9 days to about 12 days, about 12 days to about 18 days, about 12 days to about 16 days, about 12 days to about 14 days, about 14 days to about 18 days, about 14 days to about 16 days, or about 16 days to about 18 days).

In some embodiments of these methods, the liquid culture medium can be a serum-free liquid culture medium. In some embodiments of these methods, the liquid culture medium can be a chemically-defined liquid culture medium. In other examples of these methods, the liquid culture medium includes serum.

In some examples of these methods, the liquid culture medium includes the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide, and the IgGl antibody construct at a molar ratio of about 0.5: 1 to about 2: 1 (e.g., about 0.8: 1 to about 1.2: 1).

In some embodiments of these methods, the immune cell is selected from the group of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Thl7 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, gd T cell, an ab T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell. In some embodiments of any of the methods described herein, the immune cell is a T-cell or a natural killer cell.

In some examples of these methods, the immune cell was previously obtained from a subject. Some embodiments of these methods further include obtaining the immune cell from the subject prior to the contacting step. In some embodiments of these methods, the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor. Some embodiments of these methods further include, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Some

embodiments of these methods further include, before the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a

recombinant T-cell receptor. Some embodiments of these methods further include, after the contacting step, isolating the immune cell. Some embodiments of these methods further include, after the contacting step, administering the immune cell to a subject in need thereof.

In some examples of these methods, the subject has been identified or diagnosed as having an age-related disease or condition. In some examples of these methods, the age-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some examples of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples of these methods, the subject has been diagnosed or identified as having an infectious disease. In some examples of these methods, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, hepatitis C virus, papillomavirus, or influenza virus.

Also provided herein are activated immune cells produced by any of these methods. Also provided herein are pharmaceutical compositions that include any of the activated immune cells produced by any of these methods. Also provided herein are kits that include any of the pharmaceutical compositions described herein produced by any of the methods described herein.

Also provided herein are methods of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced by any of the methods described herein or any of the pharmaceutical compositions described herein.

In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some examples of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples of these methods, the subject has been identified or diagnosed as having an aging-related disease or condition. In some examples of these methods, the aging-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction

Also provided herein are methods of treating a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced by any of the methods described herein or any of the pharmaceutical compositions described herein. In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some embodiments of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,

rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some embodiments of these methods, the subject has been identified or diagnosed as having an aging-related disease or condition. In some examples of these methods, the aging-related disease or condition is selected from the group of:

Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis,

Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples of these methods, the subject has been diagnosed or identified as having an infectious disease. In some examples of these methods, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, hepatitis C virus, papillomavirus, or influenza virus. Methods of Increasing Mitochondrial Oxygen Consumption Rate of an Immune Cell

Also provided herein are methods of increasing the mitochondrial oxygen consumption rate of an immune cell that include: contacting an immune cell (e.g., any of the immune cells described herein) in a liquid culture medium (e.g., any of the liquid culture media described herein) including an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain (e.g., any of the single-chain chimeric polypeptides described herein), and optionally (ii) an IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to the linker domain (e.g., any of the exemplary IgGl antibody constructs described herein), under conditions that allow for mitochondrial oxygen consumption rate by the immune cell, where the first target- binding domain and the second target-binding domain are each independently selected from the group of: a soluble interleukin or cytokine protein, an antigen-binding domain, a soluble interleukin or cytokine receptor, and ligands of co-stimulatory molecules.

Also provided herein are methods of increasing the mitochondrial oxygen consumption rate of an immune cell that include: contacting an immune cell (e.g., any of the immune cells described herein) in a liquid culture medium (e.g., any of the liquid culture media described herein) including (1) an effective amount of a multi-chain chimeric polypeptide including: (a) a first chimeric polypeptide including: (i) a first target-binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, where the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains (e.g., any of the multi- chain chimeric polypeptides described herein); and (2) an effective amount of an IgGl antibody construct including at least one antigen-binding domain that binds specifically to the linker domain (e.g., any of the exemplary IgGl antibody constructs described herein), under conditions that allow for mitochondrial oxygen consumption rate by the immune cell.

In some embodiments of any of the methods described herein, the increase in mitochondrial oxygen consumption rate is compared to the level of mitochondrial oxygen consumption rate in a similar immune cell not contacted with the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide, and optionally the IgGl antibody construct that includes at least one antigen-binding domain that binds specifically to the soluble tissue factor domain.

Non-limiting commercial assays that can be used to detect mitochondrial oxygen consumption rate include, e.g., assays that include the use of an electrode or a

commercially available kit, e.g., Oxygen Consumption Rate Assay Kit (Cayman

Chemical), the Seahorse assay (as described in Sakamuri et ah, Geroscience 40(3):347- 356, 2018). Additional examples of assays that can be used to detect mitochondrial oxygen consumption rate are described in, e.g., Li et ah, Mitochondrial Disorders , pp. 63-72, December 2011; Kumagi et ah, Synapse e22067, August 2018; Takahashi et ah, J.

Physiol. Sci. 67(6):731-737, 2017; and Iihoshi et ah, Toxicol. Lett. 277: 109-114, 2017.

In some embodiments of these methods, the liquid culture medium includes the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide, and the IgGl antibody construct. In some examples of these methods, the IgGl antibody construct is a monoclonal IgGl antibody, where both of the antigen-binding domains in the monoclonal IgGl antibody bind specifically to the soluble tissue factor domain. In some examples of these methods, the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the soluble tissue factor domain.

In some embodiments of any of the methods described herein, the contacting step is performed for a period of about 2 hours to about 20 days (e.g., about 2 hours to about 18 days, about 2 hours to about 16 days, about 2 hours to about 14 days, about 2 hours to about 12 days, about 2 hours to about 10 days, about 2 hours to about 8 days, about 2 hours to about 7 days, about 2 hours to about 6 days, about 2 hours to about 5 days, about 2 hours to about 4 days, about 2 hours to about 3 days, about 2 hours to about 2 days, about 2 hours to about 1 day, about 6 hours to about 18 days, about 6 hours to about 16 days, about 6 hours to about 14 days, about 6 hours to about 12 days, about 6 hours to about 10 days, about 6 hours to about 8 days, about 6 hours to about 7 days, about 6 hours to about 6 days, about 6 hours to about 5 days, about 6 hours to about 4 days, about 6 hours to about 3 days, about 6 hours to about 2 days, about 6 hours to about 1 day, about 12 hours to about 18 days, about 12 hours to about 16 days, about 12 hours to about 14 days, about 12 hours to about 12 days, about 12 hours to about 10 days, about 12 hours to about 8 days, about 12 hours to about 7 days, about 12 hours to about 6 days, about 12 hours to about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12 hours to about 2 days, about 12 hours to about 1 day, about 1 day to about 18 days, about 1 day to about 16 days, about 1 day to about 15 days, about 1 day to about

14 days, about 1 day to about 12 days, about 1 day to about 10 days, about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 18 days, about 2 days to about 16 days, about 2 days to about 14 days, about 2 days to about 12 days, about 2 days to about 10 days, about 2 days to about 8 days, about 2 days to about 7 days, about 2 days to about 6 days, about 2 days to about 5 days, about 2 days to about 4 days, about 2 days to about 3 days, about 3 days to about 18 days, about 3 days to about 16 days, about 3 days to about 14 days, about 3 days to about 12 days, about 3 days to about 10 days, about 3 days to about 8 days, about 3 days to about 7 days, about 3 days to about 6 days, about 3 days to about 5 days, about 3 days to about 4 days, about 4 days to about 18 days, about 4 days to about 16 days, about 4 days to about 14 days, about 4 days to about 12 days, about 4 days to about 10 days, about 4 days to about 8 days, about 4 days to about 7 days, about 4 days to about 6 days, about 4 days to about 5 days, about 5 days to about 18 days, about 5 days to about 16 days, about 5 days to about 14 days, about 5 days to about 12 days, about 5 days to about 10 days, about 5 days to about 8 days, about 5 days to about 7 days, about 5 days to about

6 days, about 6 days to about 18 days, about 6 days to about 16 days, about 6 days to about 14 days, about 6 days to about 12 days, about 6 days to about 10 days, about 6 days to about 8 days, about 6 days to about 7 days, about 7 days to about 18 days, about 7 days to about 16 days, about 7 days to about 14 days, about 7 days to about 12 days, about 7 days to about 10 days, about 7 days to about 8 days, about 8 days to about 18 days, about 8 days to about 16 days, about 8 days to about 14 days, about 8 days to about 12 days, about 8 days to about 10 days, about 9 days to about 18 days, about 9 days to about 16 days, about 9 days to about 14 days, about 9 days to about 12 days, about 12 days to about 18 days, about 12 days to about 16 days, about 12 days to about 14 days, about 14 days to about 18 days, about 14 days to about 16 days, or about 16 days to about 18 days).

In some embodiments of these methods, the liquid culture medium can be a serum-free liquid culture medium. In some embodiments of these methods, the liquid culture medium can be a chemically-defined liquid culture medium. In other examples of these methods, the liquid culture medium includes serum.

In some examples of these methods, the liquid culture medium includes the single-chain chimeric polypeptide or the multi-chain chimeric polypeptide, and the IgGl antibody construct at a molar ratio of about 0.5: 1 to about 2: 1 (e.g., about 0.8: 1 to about 1.2: 1).

In some embodiments of these methods, the immune cell is selected from the group of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Thl7 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, gd T cell, an ab T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell. In some embodiments of any of the methods described herein, the immune cell is a T-cell or a natural killer cell.

In some examples of these methods, the immune cell was previously obtained from a subject. Some embodiments of these methods further include obtaining the immune cell from the subject prior to the contacting step. In some embodiments of these methods, the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor. Some embodiments of these methods further include, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Some

embodiments of these methods further include, before the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a

recombinant T-cell receptor. Some embodiments of these methods further include, after the contacting step, isolating the immune cell. Some embodiments of these methods further include, after the contacting step, administering the immune cell to a subject in need thereof.

In some examples of these methods, the subject has been identified or diagnosed as having an age-related disease or condition. In some examples of these methods, the age-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some examples of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples of these methods, the subject has been diagnosed or identified as having an infectious disease. In some examples of these methods, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, hepatitis C virus, papillomavirus, or influenza virus.

Also provided herein are activated immune cells produced by any of these methods. Also provided herein are pharmaceutical compositions that include any of the activated immune cells produced by any of these methods. Also provided herein are kits that include any of the pharmaceutical compositions described herein produced by any of the methods described herein.

Also provided herein are methods of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced by any of the methods described herein or any of the pharmaceutical compositions described herein.

In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some examples of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples of these methods, the subject has been identified or diagnosed as having an aging-related disease or condition. In some examples of these methods, the aging-related disease or condition is selected from the group of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction

Also provided herein are methods of treating a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced by any of the methods described herein or any of the pharmaceutical compositions described herein. In some examples of these methods, the subject has been identified or diagnosed as having a cancer. In some embodiments of these methods, the cancer is selected from the group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,

rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some embodiments of these methods, the subject has been identified or diagnosed as having an aging-related disease or condition. In some examples of these methods, the aging-related disease or condition is selected from the group of:

Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis,

Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples of these methods, the subject has been diagnosed or identified as having an infectious disease. In some examples of these methods, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, hepatitis C virus, papillomavirus, or influenza virus.

Additional Therapeutic Agents

Some embodiments of any of the methods described herein can further include administering to a subject (e.g., any of the subjects described herein) a therapeutically effective amount of one or more additional therapeutic agents. The one or more additional therapeutic agents can be administered to the subject at substantially the same time as a single-chain chimeric polypeptide (e.g., any of the single-chain chimeric polypeptides described herein), multi-chain chimeric polypeptides (e.g., any of the multi chain chimeric polypeptides described herein), or an activated immune cell (e.g., an activated NK cell or an activated T cell) (e.g., administered as a single formulation or two or more formulations to the subject). In some embodiments, one or more additional therapeutic agents can be administered to the subject prior to administration of a single- chain chimeric polypeptide (e.g., any of the single-chain chimeric polypeptides described herein), multi-chain chimeric polypeptides (e.g., any of the multi-chain chimeric polypeptides described herein), or an activated immune cell (e.g., an activated NK cell or an activated T cell). In some embodiments, one or more additional therapeutic agents can be administered to the subject after administration of a single-chain chimeric polypeptide (e.g., any of the single-chain chimeric polypeptides described herein), multi-chain chimeric polypeptides (e.g., any of the multi-chain chimeric polypeptides described herein), or an activated immune cell (e.g., an activated NK cell or an activated T cell) to the subject.

Non-limiting examples of additional therapeutic agents include: anti-cancer drugs, activating receptor agonists, immune checkpoint inhibitors, agents for blocking HLA- specific inhibitory receptors, Glucogen Synthase Kinase (GSK) 3 inhibitors, and antibodies.

Non-limiting examples of anticancer drugs include antimetabolic drugs (e.g., 5- fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxycarbamide, methotrexate, 6-thioguanine, cladribine, nelarabine, pentostatin, or pemetrexed), plant alkaloids (e.g., vinblastine, vincristine, vindesine, camptothecin, 9-methoxycamptothecin, coronaridine, taxol, naucleaorals, diprenylated indole alkaloid, montamine, schischkiniin, protoberberine, berberine, sanguinarine, chelerythrine, chelidonine, liriodenine, clivorine, b-carboline, antofme, tylophorine, cryptolepine, neocryptolepine, corynoline, sampangine, carbazole, crinamine, montanine, ellipticine, paclitaxel, docetaxel, etoposide, tenisopide, irinotecan, topotecan, or acridone alkaloids), proteasome inhibitors (e.g., lactacystin, disulfiram, epigallocatechin-3-gallate, marizomib (salinosporamide A), oprozomib (ONX-0912), delanzomib (CEP- 18770), epoxomicin, MG132, beta-hydroxy beta-methylbutyrate, bortezomib, carfilzomib, or ixazomib), antitumor antibiotics (e.g., doxorubicin, daunorubicin, epirubicin, mitoxantrone, idarubicin, actinomycin, plicamycin, mitomycin, or bleomycin), histone deacetylase inhibitors (e.g., vorinostat, panobinostat, belinostat, givinostat, abexinostat, depsipeptide, entinostat, phenyl butyrate, valproic acid,

trichostatin A, dacinostat, mocetinostat, pracinostat, nicotinamide, cambinol, tenovin 1, tenovin 6, sirtinol, ricolinostat, tefmostat, kevetrin, quisinostat, resminostat, tacedinaline, chidamide, or selisistat), tyrosine kinase inhibitors (e.g., axitinib, dasatinib, encorafmib, erlotinib, imatinib, nilotinib, pazopanib, and sunitinib), and chemotherapeutic agents (e.g., all-trans retinoic acid, azacitidine, azathioprine, doxifluridine, epothilone,

hydroxyurea, imatinib, teniposide, tioguanine, valrubicin, vemurafenib, and

lenalidomide). Additional examples of chemotherapeutic agents include alkylating agents, e.g., mechlorethamine, cyclophosphamide, chlorambucil, melphalan, ifosfamide, thiotepa, hexamethylmelamine, busulfan, altretamine, procarbazine, dacarbazine, temozolomide, carmustine, lumustine, streptozocin, carboplatin, cisplatin, and

oxaliplatin.

Non-limiting examples of activating receptor agonists include any agonists for activating receptors which activate and enhance the cytotoxicity of NK cells, including anti-CDl6 antibodies (e.g., anti-CDl6/CD30 bispecific monoclonal antibody (BiMAb)) and Fc-based fusion proteins. Non-limiting examples of checkpoint inhibitors include anti-PD-l antibodies (e.g., MEDI0680), anti-PD-Ll antibodies (e.g., BCD-135, BGB- A333, CBT-502, CK-301, CS1001, FAZ053, KN035, MDX-1105, MSB2311, SHR- 1316, anti-PD-Ll/CTLA-4 bispecific antibody KN046, anti-PD-L l /TGFpRII fusion protein M7824, anti-PD-Ll/TIM-3 bispecific antibody LY3415244, atezolizumab, or avelumab), anti-TIM3 antibodies (e.g., TSR-022, Sym023, or MBG453) and anti-CTLA- 4 antibodies (e.g., AGEN1884, MK-1308, or an anti-CTLA-4/OX40 bispecific antibody

ATOR-1015). Non-limiting examples of agents for blocking HLA-specific inhibitory receptors include monalizumab (e.g., an anti-HLA-E NKG2A inhibitory receptor monoclonal antibody). Non-limiting examples of GSK3 inhibitor include tideglusib or CHIR99021. Non-limiting examples of antibodies that can be used as additional therapeutic agents include anti-CD26 antibodies (e.g., YS110), anti-CD36 antibodies, and any other antibody or antibody construct that can bind to and activate an Fc receptor (e.g., CD 16) on a NK cell. In some embodiments, an additional therapeutic agent can be insulin or metformin. EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1. Construction of exemplary multi-chain chimeric polypeptides and evaluation of properties thereof

Two multi-chain chimeric polypeptides were generated and their properties were evaluated. Each of the two multi-chain chimeric polypeptides included a first chimeric polypeptide that included a soluble tissue factor domain covalently linked a first target- binding domain and a first domain of an affinity pair of domains. The second chimeric polypeptide in each of the two multi-chain chimeric polypeptides included a second domain of the affinity pair of domains, and a second target-binding domain.

Description of logic underlying construction of multi-chain chimeric polypeptides

Tissue Factor (TF) is a stable, transmembrane protein containing 236 amino acid residues. The truncated, recombinant 219-amino-acid extracellular domain of tissue factor is soluble and is known to be expressed at high levels in bacteria or mammalian cells. Without wishing to be bound to a particular theory, the applicants speculated that the 2l9-aa tissue factor could be used as a connector linker for creation of unique multi- chain chimeric polypeptides.

First chimeric polypeptides including soluble tissue factor domain were produced at high levels by CHO-K1 cells grown in fermentation broth. These first chimeric polypeptides were purified by an anti-tissue factor monoclonal antibody (mAh) coupled on a solid matrix. Notably, tissue factor contains binding sites for FVIIa and FX. The catalytic activity of the tissue factor-FVIIa complex for FX is approximately 1 million- fold lower when tissue factor is not anchored to a phospholipid bilayer. Thus, without wishing to be bound to a particular theory, applicants speculated that using the 2l9-aa extracellular domain of tissue factor without the transmembrane in construction of the first chimeric polypeptides may eliminate the pro-coagulation activity of tissue factor in the first chimeric polypeptides. In an effort to further reduce or eliminate the pro- coagulation activity of the 2l9-aa tissue factor, select mutations in tissue factor can be made, specifically at seven amino acid residues that are known to contribute to binding energy of the FVIIa binding site.

Characterization of binding interactions for described chimeric polypeptides To determine if the first and second chimeric polypeptides bind to each other to form multi-chain chimeric polypeptides, in vitro binding assays were performed. To determine if the first chimeric polypeptide comprising soluble tissue factor domain are recognized and bound by anti-TF mAh, in vitro binding assays were performed. Notably, the data indicated that the mutated tissue factor proteins are still recognized and selectively bound by the anti-TF mAh which is known to bind to the FX binding site on tissue factor. To determine if the first chimeric polypeptides comprising soluble tissue factor domain covalently linked to scFvs or cytokines (see Figure 1 and Figure 2) possess functional scFvs or cytokines, in vitro binding assays were performed. The data from the aforementioned assays were consistent with the purified first chimeric polypeptides having the expected biological activities (e.g., scFvs selectively bind expected target antigens or cytokines selectively bind expected receptors or binding proteins).

In addition, experiments performed using the two multi-chain chimeric polypeptides including a first and second chimeric polypeptide bound to each other demonstrate the expected target binding activity (e.g., the multi-chain chimeric polypeptide binds specifically to the target specifically recognized by the first target- binding domain and the target specifically recognized by the second target-binding domain).

Based on the aforementioned results, applicants concluded that the soluble tissue factor connecter linker provided or enabled appropriate display of the polypeptides encoding either scFvs, interleukins, cytokines, interleukin receptors, or cytokine receptors in three-dimensional space relative to soluble tissue factor domain and relative to one another such that each retained expected biological properties (and activities).

When both the first and second chimeric polypeptides were co-expressed, the heterodimeric complexes were secreted into the fermentation broths at high levels. The complexes were captured and readily purified by anti-TF mAh conjugated to a solid matrix using affinity chromatography. The first and second target-binding domains of these multi-chain chimeric polypeptides retained their expected biological activities as assayed by in vitro binding assays. Thus, the assembly of the multi-chain chimeric polypeptides provides the appropriate spatial display and folding of the domains for biological activities. Importantly, the spatial arrangement of the multi-chain chimeric polypeptides does not interfere with the FX binding site on tissue factor which enables the use of anti-TF mAh for affinity purification.

Characterization of stability for described chimeric polypeptides

Both purified multi-chain chimeric polypeptides are expected to be stable. These multi-chain chimeric polypeptides are structurally intact and expected to be fully biologically active when they are incubated in human serum at 37 °C for 72 hours.

Characterization of propensity of described chimeric polypeptides to aggregate

Both purified multi-chain chimeric polypeptides are expected not to form aggregates when stored at 4 °C in PBS.

Characterization of viscosity of described chimeric polypeptides

Compositions including the multi-chain chimeric polypeptides are not expected to have any viscosity issues when formulated at a concentration as high as 50 mg/mL in PBS.

Discussion of multi-chain chimeric polypeptide platform for use in selectively binding clinically relevant targets

The data from our studies show that our platform technologies can be utilized to create molecules that could be fused to target-binding domains derived from antibodies, in any formats as discussed above, adhesion molecules, receptors, cytokines, chemokines etc. With the appropriate target-binding domain, the resulting multi-chain chimeric polypeptides could promote conjugation of various immune effector cells and mediate destruction of target cells, including cancer cells, virally-infected cells, or senescent cells. Other domains in the multi-chain chimeric polypeptides stimulate, activate, and attract the immune system for enhancing cytotoxicity of effector cells for the targeted cells. Example 2: Creation of an IL-7/IL-15RaSu DNA construct

In a non-limiting example, an IL-7/IL-l5RaSu DNA construct was created (see Figure 1). The human IL-7 sequence, human IL-l5RaSu sequence, human IL-15 sequence, and human tissue factor 219 sequence were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the IL-7 sequence to the IL-l5RaSu sequence. The final IL-7/IL-l5RaSu DNA construct sequence was synthesized by Genewiz.

Example 3: Creation of an IL-21/TF/IL-15 DNA construct

In a non-limiting example, an IL-21/TF/IL-15 construct was made (see Figure 2) by linking the IL-21 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-21/TF construct with the N-terminus coding region of IL-15.

Example 4: Secretion of IL-7/IL-15RaSu and IL-21/TF/IL-15 fusion proteins

The IL-7/IL-l5RaSu and IL-21/TF/IL-15 DNA constructs were cloned into a pMSGV-l modified retrovirus expression vector (as described by Hughes, Hum Gene Ther 16:457-72, 2005, hereby incorporated by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of a soluble IL-2l/TF/IL-l5:IL-7/IL-l5RaSu protein complex (referred to as 2ltl5-7s, see Figures 3 and 4). The 2ltl5-7s protein was purified from CHO-K1 cell culture supernatant using anti-TF antibody affinity chromatography and size exclusion chromatography resulting in soluble (non-aggregated) protein complexes consisting of IL-7/IL-l5RaSu and IL-21/TF/IL-15 fusion proteins.

In some cases, the leader (signal sequence) peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

Example 5: Purification of 21tl5-7s by immunoaffinity chromatography

An anti-TF antibody affinity column was connected to a GE Healthcare™ AKTA

Avant protein purification system. The flow rate was 4 mL/min for all steps except the elution step, which was 2 mL/min.

Cell culture harvest of 2ltl5-7s was adjusted to pH 7.4 with 1M Tris base and loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes PBS, followed by elution with 6 column volumes 0.1M acetic acid, pH 2.9. Absorbance at 280 nm was collected and then the sample was neutralized to pH 7.5-8.0 by adding 1M Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon® centrifugal filters with a 30 KDa molecular weight cutoff. The buffer-exchanged protein sample was stored at 2-8°C for further biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was then stripped using 6 column volumes 0.1M glycine, pH 2.5. The column was then neutralized using 10 column volumes PBS, 0.05% sodium azide and stored at 2-8°C.

Example 6: Creation of a TGF-pRII /IL-15RaSu DNA construct

In a non-limiting example, a TGF-PRII/IL- 15RaSu DNA construct was created (see Figure 8). The human TGF-PRII sequence, human IL-l5RaSu sequence, human IL- 15 sequence, and human tissue factor 219 sequence were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the TGF-PRII sequences (separated by a linker) to the IL-l5RaSu sequence. The final TGF-PRII /IL-l5RaSu DNA construct sequence was synthesized by Genewiz.

Example 7: Creation of an IL-21/TF/IL-15 DNA construct

In a non-limiting example, an IL-21/TF/IL-15 construct was made (see Figure 9) by linking the IL-21 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-21/TF construct with the N-terminus coding region of IL-15.

Example 8: Secretion of TGF-pRII /IL-15RaSu and IL-21/TF/IL-15 fusion proteins

The TGF-PRII /IL-l5RaSu and IL-21/TF/IL-15 DNA constructs were cloned into a pMSGV-l modified retrovirus expression vector (as described by Hughes, Hum Gene Ther 16:457-72, 2005, hereby incorporated by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of a soluble IL-21/TF/IL-15: TGF-b RII/IL-l5RaSu protein complex (referred to as 2ltl5-TGFRs, see Figures 10 and 11). The 2ltl5-TGFRs protein was purified from CHO-K1 cell culture supernatant using anti-TF antibody affinity chromatography and size exclusion chromatography resulting in soluble (non- aggregated) protein complexes consisting of TGF-b RII/IL- 15RaSu and IL-21/TF/IL-15 fusion proteins. Example 9: Size exclusion chromatography

A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration column was connected to a GE Healthcare AKTA™ Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. A capillary loop was used to inject 200pL of 1 mg/mL of 7tl5-2lscomplex onto the column. The injection was chased with 1.25 column volumes of PBS.

Example 10: SDS-PAGE of 21tl5-7s and 21tl5-TGFRs

To determine the purity and protein molecular weight, the purified 2ltl5-7s or 2ltl5-TGFRs protein sample were analyzed using 4-12% NuPage Bis-Tris protein gel SDS-PAGE. The gel will be stained with InstantBlue™ for about 30 min, followed by destaining overnight in purified water.

Example 11: Glycosylation of 21tl5-7s and 21tl5-TGFRs in CHO-K1 cells

Glycosylation of 2 ltl 5-7s in CHO-K1 cells or 2ltl5-TGFRs in CHO-K1 cells were confirmed using the Protein Deglycosylation Mix II kit (New England Biolabs), according to the manufacturer’s instructions. Example 12: Recombinant protein quantitation of 21tl5-7s and 21tl5-TGFRs complexes

The 2ltl5-7s complex or the 2ltl5-TGFRs complex were detected and quantified using standard sandwich ELISA methods. Anti-human tissue factor antibody (IgGl) served as the capture antibody and biotinylated anti-human IL-21, IL-15, or IL-7 antibody (2ltl5-7s) or biotinylated anti-human IL-21, IL-15, or TGF-PRII antibody (2ltl5-TGFRs) served as the detection antibody. Tissue factor in purified 2ltl5-7s or 2ltl5-TGFRs protein complexes was detected using an anti -human tissue factor capture antibody, and anti-human tissue factor antibody (IgGl) detection antibody. The anti-TF ELISA will be compared to purified tissue factor at similar concentrations.

Example 13: Expansion capacity of primary natural killer (NK) cells by 21tl5-7s complex + anti-TF IgGl antibody or 21tl5-TGFRs complex + anti-TF IgGl antibody

To assess the 2ltl5-7s complex’s ability to expand primary natural killer (NK) cells, 2ltl5-7s complex and 2ltl5-7s complex + anti-TF IgGl antibody was added to NK cells obtained from samples of fresh human leukocytes. Cells were stimulated with 50nM of 2 ltl 5-7s complex with or without 25 nM of anti-TF IgGl or anti-TF IgG4 antibody at 37° and 5% CO2. Cells were maintained at concentration at 0.5 x l0 ⁶ /mL not exceeding 2.0 x l0 ⁶ /mL by counting every 48-72 hours and media was replenished with fresh stimulator. Cells stimulated with 2ltl5-7s complex or anti-TF IgGl antibody or anti-TF IgG4 antibody or anti-TF IgG4 + 2ltl5-7s complex were maintained up to day 5. Figure 5 shows expansion of primary NK cells upon incubation with 2ltl5-7s complex + anti-TF IgGl antibody.

To assess the 2ltl5-TGFRs complex’s ability to expand primary natural killer (NK) cells, 2ltl5-TGFRs complex and 2ltl5-TGFRs complex + anti-TF IgGl antibody was added to NK cells obtained from samples of fresh human leukocytes. Cells were stimulated with 50nM of 2ltl5-TGFRs complex with or without 25 nM of anti-TF IgGl or anti-TF IgG4 antibody at 37°C and 5% CO2. Cells were maintained at concentration at 0.5 x l0 ⁶ /mL not exceeding 2.0 x l0 ⁶ /mL by counting every 48-72 hours and media was replenished with fresh stimulator. Cells stimulated with 2ltl5-TGFRs complex or anti- TF IgGl antibody or anti-TF IgG4 antibody or anti-TF IgG4 + 2ltl5-TGFRs complex were maintained up to day 5. Figure 12 shows expansion of primary NK cells upon incubation with 2ltl5-TGFRs complex + anti-TF IgGl antibody. Figure 18 is a schematic summarizing these results. Example 14: Activation of expanded NK cells by the 21tl5-7s complex + anti-TF IgGl antibody or the 21tl5-TGFRs complex + anti-TF IgGl antibody

Primary NK cells can be induced ex vivo following overnight stimulation of purified NK cells with 2ltl5-7s complex + anti-TF IgGl antibody. Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >80% and confirmed by staining with CD56-BV421 and CD16-BV510 specific antibodies (BioLegend). Cells were counted and resuspended in 1 x l0 ⁶ /mL in a 24 well flat bottom plate in 1 mL of complete media (RPMI 1640 (Gibco), supplemented with 4 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), non-essential amino acid (Thermo Life Technologies), sodium pyruvate (Thermo Life Technologies), and 10% FBS (Hyclone)). Cells were stimulated with 50 nM of 2ltl5-7s with or without 25 nM of TF IgGl antibody at 37°C and 5% CO2. Cells were counted every 48-72 hours and maintained at a concentration of 0.5 x l0 ⁶ /mL to 2.0 x l0 ⁶ /mL until dayl4. Media was periodically replenished with fresh stimulator. Cells were harvested and surface stained at day 3 with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies

(Biolegend and analyzed by Flow Cytometry-Celeste-BD Bioscience). Figure 6 shows the activation markers CD25 MFI and CD69 MFI. The activation marker CD25 MFI increased with 2ltl5-7s complex + anti-TF IgGl antibody stimulation, but not 2ltl5-7s complex stimulation. The activation marker CD69 MFI increased with both 2 ltl 5-7s complex + anti-TF IgGl antibody and with 2ltl5-7s complex, alone.

Primary NK cells can be induced ex vivo following overnight stimulation of purified NK cells with 2ltl5-TGFRs complex + anti-TF IgGl antibody. Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >80% and confirmed by staining with CD56-BV421 and CD16-BV510 specific antibodies (BioLegend). Cells were counted and resuspended in 1 x l0 ⁶ /mL in a 24 well flat bottom plate in 1 mL of complete media (RPMI 1640 (Gibco), supplemented with 4 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), non-essential amino acid (Thermo Life Technologies), sodium pyruvate (Thermo Life Technologies), and 10% FBS (Hyclone)). Cells were stimulated with 50 nM of 2ltl5-TGFRs with or without 25 nM of anti-TF IgGl antibody at 37°C and 5% CO2. Cells were counted every 48-72 hours and maintained at a concentration of 0.5 x l0 ⁶ /mL to 2.0 x l0 ⁶ /mL until dayl4. Media was periodically replenished with fresh stimulator. Cells were harvested and surface stained at day 3 with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies (Biolegend and analyzed by Flow Cytometry-Celeste-BD Bioscience). Figure 13 shows the activation markers CD25 MFI and CD69 MFI. The activation marker CD25 MFI increased with 2ltl5-TGFRs complex + anti-TF IgGl antibody stimulation, but not 2ltl5-TGFRs complex stimulation. The activation marker CD69 MFI increased with both 2ltl5-TGFRs complex + anti-TF IgGl antibody and with 2ltl5-TGFRs complex, alone.

Example 15: Cytotoxicity of NK cells against human tumor cells

Fresh human leukocytes were obtained from a blood bank. NK cells were isolated via negative selection using the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >80% and confirmed by staining with CD56- BV421 and CD16-BV510 specific antibodies (BioLegend). Cells were counted and resuspended in 1 x l0 ⁶ /mL in a 24 well, flat bottom plate in 1 mL of complete media (RPMI 1640 (Gibco)), supplemented with 4 mM L-glutamine (Thermo Life

Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life

Technologies), non-essential amino acid (Thermo Life Technologies), sodium pyruvate (Thermo Life Technologies) and 10% FBS (Hyclone). Cells were stimulated with 21U5- 7s 50 nM and 25 nM of anti-TF IgGl antibody for up to 14 days at 37°C and 5% CO2. Cells were counted every 48-72 hours and maintained at a concentration of 0.5 x l0 ⁶ /mL to 2.0 x l0 ⁶ /mL until dayl4. Media was periodically replenished with fresh stimulator. On day 14, cells were harvested and washed 2 times. K562 human tumor cells were harvested and labeled with Celltrace violet. Celltrace violet-labeled K562 cells were mixed with the in vitro expanded NK cells and incubated at 37°C for 20 hours. The mixture was harvested and the percentage of dead K562 cells were determined by propidium iodide staining and flow cytometry. Figure 7 shows increased specific lysis of K562 cells when incubated with expanded NK cells.

Example 16: Creation of an IL-21/IL-15RaSu DNA construct

In a non-limiting example, an IL-2l/IL-l5RaSu DNA construct was created. The human IL-21 sequence and human IL-l5RaSu sequence were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the IL-21 sequence to the IL-l5RaSu sequence. The final IL-21/IL- l5RaSu DNA construct sequence was synthesized by Genewiz. See Figure 14.

Example 17: Creation of an IL-7/TF/IL-15 DNA construct

In a non-limiting example, an IL-7/TF/IL-15 construct was made by linking the

IL-7 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-7/TF construct with the N-terminus coding region of IL-15. See Figure 15.

Example 18: Creation of an IL-21/IL-15Ra Sushi DNA construct

In a non-limiting example, a second chimeric polypeptide of IL-2l/IL-l5RaSu was generated. The human IL-21 and human IL-l5Ra sushi sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the IL-21 sequence to the IL-l5Ra sushi sequence.

The final IL-2l/IL-l5RaSu DNA construct sequence was synthesized by Genewiz.

Example 19: Creation of an IL-7/TF/IL-15 DNA construct

In a non-limiting example, an exemplary first chimeric polypeptide of IL-

7/TF/IL-15 was made by linking the IL-7 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-7/TF construct with the N-terminus coding region of IL-15. The nucleic acid sequence encoding the first chimeric polypeptide of IL-7/TF/IL-15 (including leader sequence), synthesized by Genewiz. Example 20: Secretion of IL-21/IL-15RaSu and IL-7/TF/IL-15 fusion proteins

The IL-2l/IL-l5RaSu and IL-7/TF/IL-15 DNA constructs were cloned into a pMSGV-l modified retrovirus expression vector (as described by Hughes, Hum Gene Ther 16:457-72, 2005, hereby incorporated by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of a soluble IL-7/TF/IL-l5:IL-2l/IL-l5RaSu protein complex (referred to as 7tl5-2ls). The 7tl 5-21 s protein was purified from CHO-K1 cell culture supernatant using anti-TF antibody (IgGl) affinity chromatography and size exclusion chromatography resulting in soluble (non-aggregated) protein complexes consisting of IL-2l/IL-l5RaSu and IL-7/TF/IL-15 fusion proteins. See, Figures 16 and 17.

Example 21: Analytical size exclusion chromatography (SEC) analysis of IL-21/IL- 15RaSu and IL-7/TF/IL-15 fusion proteins

To determine if anti-tissue factor monoclonal antibody and 7tl 5-21 s can form an antibody-fusion-molecule complex, analytical size exclusion chromatography (SEC) was performed. A Superdex 200 Increase 10/300 GL gel filtration column (from GE

Healthcare) was connected to an AKTA Avant system (from GE Healthcare). The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. Samples of the anti-TF mAh (1 mg/mL), 7tl5-2ls (lmg/mL), and a mixture of combined at a 1 : 1 ratio, so the final concentration of each protein is 0.5mg/mL) were in PBS. Each sample was injected into the Superdex 200 column using a capillary loop, and analyzed by SEC. The SEC chromatograph of each sample was shown in Figure 19. The SEC results indicated that there are two protein peaks for 7tl 5-21 s, likely representing a dimer (with an apparent molecular weight of l99.2kDa) and a higher oligomer of 7tl 5-21 s, and there is one peak (with an apparent molecular weight of 206.8kDa) for the anti-TF mAh. However, as expected, a new protein peak with a higher molecular weight (with an apparent molecular weight of 576.9 kDa) was formed in the mixture sample containing the anti-TF mAh and 7tl 5-21 s, indicating that the anti-TF mAh and 7tl 5-21 s form an antibody-antigen complex through the binding of anti-TF mAh to TF in the fusion protein complex. Example 22: Expansion capacity of primary natural killer (NK) cells by 7tl5-21s complex + anti-TF IgGl antibody

To assess the 7tl 5-21 s complex’s ability to expand primary natural killer (NK) cells, 7tl5-2ls complex and 7tl5-2ls complex + anti-TF IgGl antibody are added to K cells obtained from samples of fresh human leukocytes. Cells are stimulated with 50nM of 7tl 5-21 s complex with or without 25 nM of anti-TF IgGl or anti-TF IgG4 antibody at 37°C and 5% CO2. Cells are maintained at concentration at 0.5 x l0 ⁶ /mL not exceeding 2.0 x l0 ⁶ /mL by counting every 48-72 hours and media is replenished with fresh stimulator. Cells stimulated with 7tl5-2ls complex or anti-TF IgGl antibody or anti-TF IgG4 antibody or anti-TF IgG4 + 7tl 5-21 s complex are maintained up to day 5.

Expansion of primary NK cells upon incubation with 2ltl5-7s complex + anti-TF IgGl antibody is observed.

Example 23: Activation of expanded NK cells by the 7tl5-21s complex + anti-TF IgGl antibody

Primary NK cells are induced ex vivo following overnight stimulation of purified

NK cells with 7tl5-2ls complex + TF IgGl antibody. Fresh human leukocytes are obtained from a blood bank and CD56+ NK cells are isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells is >80% and is confirmed by staining with CD56-BV421 and CD16-BV510 specific antibodies

(BioLegend). Cells are counted and resuspended in 1 x l0 ⁶ /mL in a 24 well flat bottom plate in 1 mL of complete media (RPMI 1640 (Gibco), supplemented with 4 mM L- glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), non-essential amino acid (Thermo Life Technologies), sodium pyruvate (Thermo Life Technologies), and 10% FBS (Hyclone)). Cells are stimulated with 50 nM of 7tl 5-21 s with or without 25 nM of anti-TF IgGl antibody at 37°C and 5% CO2. Cells are counted every 48-72 hours and maintained at a concentration of 0.5 x l0 ⁶ /mL to 2.0 x l0 ⁶ /mL until day 14. Media is periodically replenished with fresh stimulator. Cells are harvested and surface stained at day 3 with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies (Biolegend and analyzed by Flow Cytometry-Celeste-BD Bioscience). The activation marker CD25 MFI are observed to increase with 7tl 5-21 s complex + anti-TF IgGl antibody stimulation, but not 7tl5-2ls complex stimulation. The activation marker CD69 MFI is observed to increase with both 7tl 5-21 s complex + anti-TF IgGl antibody and with 7tl 5-21 s complex, alone. Example 24: Increase in Glucose Metabolism in NK cells Using 7tl5-21s and 21tl5- 7s in Combination with an Anti-TF IgGl Antibody

A set of experiments was performed to determine the effect of (1) 7t 15-21 s in combination with an anti-TF IgGl antibody or (2) 2 ltl 5-7s in combination with an anti- TF IgGl antibody on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) on NK cells purified from human blood. In these experiments, fresh human leukocytes were obtained from the blood bank and NK cells were isolated via negative selection using the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >80% and confirmed by staining with CD56-BV421 and CD16- BV510 specific antibodies (BioLegend). The cells were counted and resuspended at a concentration of 1 x l0 ⁶ /mL in a 24-well flat-bottom plate in 1 mL of complete media (RPMI 1640 (Gibco) supplemented with 4 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), non- essential amino acid (Thermo Life Technologies), sodium pyruvate (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were stimulated with (1) 100 nM anti-TF IgGl antibody, (2) 50 nM 7tl 5-21 s and 100 nM anti-TF IgGl antibody, or (3) 50 nM 2ltl5-7s and 100 nM anti-TF IgGl, for up to day 5 at 37 °C, 5% CO2. The cells were maintained at concentration at 0.5 x l0 ⁶ /mL not exceeding 2.0 x l0 ⁶ /mL until day 5 by counting every 48 hours and media was replenished with fresh stimulator. Extracellular flux assays on expanded NK cells were performed using a XFp Analyzer (Seahorse Bioscience). The harvested cells were washed and plated at 2.0 x 10 ⁵ cells/well in at least duplicate for extracellular flux analysis of OCR (Oxygen Consumption Rate) and ECAR (Extracellular Acidification Rate). Glycolysis stress tests were performed in Seahorse Media containing 2 mM of glutamine. The following were used during the assay: 10 mM glucose; 100 nM oligomycin; and 100 mM 2-deoxy-D-glycose (2DG). The data show that the combinations of (1) 100 nM 7tl 5-21 s and 50 nM anti-TF IgGl antibody, and (2) 100 nM 2ltl5-7s and 50 nM anti-TF IgGl result in a significant increase in oxygen consumption rate (Figure 20) and extracellular acidification rate (ECAR) (Figure 21). Example 25: Increase in Glucose Metabolism in NK Cells Using 18tl5-12s

A set of experiments was performed to determine the effect of the construct of l8tl5-l2s (Figure 22) on oxygen consumption rate and extracellular acidification rate (ECAR) on NK cells purified from human blood.

In these experiments, fresh human leukocytes were obtained from the blood bank from two different human donors and NK cells were isolated via negative selection using the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >80% and confirmed by staining with CD56-BV421 and CD16-BV510 specific antibodies (BioLegend). The cells were counted and resuspended in 2 x l0 ⁶ /mL in 24- well, flat-bottom plates in 1 mL of complete media (RPMI 1640 (Gibco) supplemented with 4 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life

Technologies), streptomycin (Thermo Life Technologies), non-essential amino acid (Thermo Life Technologies), sodium pyruvate (Thermo Life Technologies) and 10%

FBS (Hyclone)). The cells were stimulated with either (1) media alone, (2) 100 nM l8tl5-l2s, or (3) mixture of single cytokines recombinant human IL-12 (0.25 pg), recombinant human IL-15 (1.25 pg), and recombinant human IL-18 (1.25 pg) overnight at 37 °C, 5% CO2. On the next day, the cells were harvested and extracellular flux assays on expanded NK cells were performed using a XFp Analyzer (Seahorse Bioscience). The harvested cells washed and plated 2.0 x 10 ⁵ cells/well in at least duplicate for

extracellular flux analysis of OCR (Oxygen Consumption Rate) and ECAR (Extracellular Acidification Rate). The glycolysis stress tests were performed in Seahorse Media contain 2 mM of glutamine. The following were used during the assay: 10 mM glucose; 100 nM oligomycin; and 100 mM 2-deoxy-D-glycose (2DG).

The data show that the l8tl5-l2s results in significantly increased oxygen consumption rate (Figure 23) and extracellular acidification rate (ECAR) as compared to the same cells activated with a combination of recombinant human IL-12, recombinant human IL-15, and recombinant human IL-18 (Figure 24).

Example 26: Creation of an IL-12/IL-15RaSu DNA construct

In a non-limiting example, an IL-l2/IL-l5RaSu DNA construct was created. (see Figure 25) The human IL-12 subunit sequences, human IL-l5RaSu sequence, human IL-15 sequence, human tissue factor 219 sequence, and human IL-18 sequence were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the IL-12 subunit beta (p40) to IL-12 subunit alpha (p35) with a GS (3) linker to generate a single chain version of IL-12 and then directly linking the IL-12 sequence to the IL-l5RaSu sequence. The final IL-12/IL- l5RaSu DNA construct sequence was synthesized by Genewiz.

The nucleic acid sequence of the åLl2/IL-l5RaSu construct (including signal peptide sequence) is as follows (SEQ ID NO: 123):

(Signal peptide)

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCT

ACTCC

(Human IL-12 subunit beta (p40))

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTAT CCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAG ACGGC AT C ACTTGGACCCTCGAT C AGAGC AGCGAGGT GCTGGGCTCCGGAAA GACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGC CACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGG AAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGA ATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGT TGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCG GGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCT GAGAGGGTTCGT GGCGAT AAC AAGGAAT ACGAGT AC AGCGTGGAGT GCC AA GAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGG TGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATC CGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCA A A A AT AGCC GGC A AGTTGAGGT C TC TT GGGA AT ATCCC GAC AC TT GGAGC AC ACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCA AGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCA TCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCC AGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC

{Linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

{Human IL-12 subunit alpha (p35))

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTAC ACC ACAGCCAGAATTTACTGAGGGCCGTGAGC AACATGCTGC AGAAAGCTAG GCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGAT ATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGA CAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGG CTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTC CATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCC AAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGG CTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCT CAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGT GCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATG AGCTATTTAAACGCCAGC

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

Example 27: Creation of an IL-18/TF/IL-15 DNA construct

In a non-limiting example, an IL-18/TF/IL-15 construct was made (see Figure 26) linking the IL-18 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-18/TF construct with the N-terminus coding region of IL-15. The nucleic acid sequence of the IL-18/TF/IL-15 construct (including leader sequence), synthesized by Genewiz, is as follows (SEQ ID NO: 1 19):

(Signal peptide)

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCT

ACAGC

(Human IL-18)

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAC GACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGAC CGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGT ACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGA GAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATG AACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGC GGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGA GGGC T AC TTTTT AGC TT GT GA A A AGGAGAGGGATTT ATT C A AGC T GATCC T C A AGAAGGAGGACGAGCTGGGCGATCGTTCC AT CAT GTT C ACCGTCC AA AACGA GGAT

(Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG (Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

Example 28: Secretion of IL-12/IL-15RaSu and IL-18/TF/IL-15 fusion proteins The IL- 12/IL- 15RaSu and IL- 18/TF/IL- 15 DNA constructs were cloned into a pMSGV-l modified retrovirus expression vector (as described by Hughes, Hum Gene Hier 16:457-72, 2005, hereby incorporated by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of a soluble IL-l 8/TF/IL- 15 :IL-l 2/IL- l5RaSu protein complex (referred to as 18tl 5-l2s, see Figures 27 and 28). The 18tl 5-l2s protein was purified from CHO-K1 cell culture supernatant using anti-TF antibody affinity chromatography and size exclusion chromatography resulting in soluble (non-aggregated) protein complexes consisting of IL-l 2/IL- l5RaSu and IL-l 8/TF/IL- 15 fusion proteins.

The amino acid sequence of the ILl2/IL-l5RaSu fusion protein (including signal peptide sequence) is as follows (SEQ ID NO: 122):

(Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-l 2 subunit beta (p40))

IWELKKD VYVVELDWYPD APGEMVVLT CDTPEEDGITWTLDQ S SEVLGS

GKTLTIQ VKEF GD AGQ YTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKN KTFLRCEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQGVTCGAATLS AERV RGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPD PPKNLQLKPLKN SRQVEV SWEYPDTW STPHS YF SLTF C VQ VQGKSKREKKDRVF TDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS {Linker)

GGGGS GGGGS GGGGS

{Human IL-12 subunit alpha (p35))

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHE DITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYE DLKMY QVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQ ALNFN SETVPQKS S LEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNKAT NVAHWTTPSLKCIR

The amino acid sequence of the IL-18/TF/IL-15 fusion protein (including signal peptide sequence) is as follows (SEQ ID NO: 118):

(Signal peptide)

MKWVTFISLLFLF S SAY S

(Human IL-18)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISM YKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPG HDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS In some cases, the leader (signal sequence) peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

Example 29: Purification of 18tl5-12s by immunoaffinity chromatography

An anti-TF antibody affinity column was connected to a GE Healthcare™ AKTA Avant protein purification system. The flow rate was 4 mL/min for all steps except the elution step, which was 2 mL/min.

Cell culture harvest of l8tl5-l2s was adjusted to pH 7.4 with 1M Tris base and loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes PBS, followed by elution with 6 column volumes 0.1M acetic acid, pH 2.9. Absorbance at 280 nm was collected and then the sample was neutralized to pH 7.5-8.0 by adding 1M Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon® centrifugal filters with a 30 KDa molecular weight cutoff. Figure 29 shows that the l8tl5-l2s complex binds the anti-TF antibody affinity column, wherein TF is an 18tl 5- l2s binding partner. The buffer-exchanged protein sample is stored at 2-8°C for further biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was then stripped using 6 column volumes 0.1M glycine, pH 2.5. The column was then neutralized using 10 column volumes PBS, 0.05% sodium azide and stored at 2-8°C. Example 30: Size exclusion chromatography of 18tl5-12s

A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration column was connected to a GE Healthcare AKTA™ Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.8 mL/min. A capillary loop was used to inject 200pL of 1 mg/mL of l8tl5-l2s complex onto the column. The injection was chased with 1.25 column volumes of PBS. The SEC chromatograph is shown in Figure 30. There is a main 18U5-12S protein peak with a minor high molecular weight peak, likely due to differing degrees of glycosylation of l8tl5-l2s dimers or aggregates. Example 31: SDS-PAGE of 18tl5-12s

To determine the purity and protein molecular weight, the purified l8tl5-l2s protein sample was analyzed using 4-12% NuPage Bis-Tris protein gel SDS-PAGE. The gel was stained with InstantBlue™ for about 30 min, followed by destaining overnight in purified water. Figure 31 shows an example SDS gel of anti-TF antibody affinity purified 18tl 5-l2s, with bands at the expected molecular weights (66 kDa and 56 kDa).

Example 32: Glycosylation of 18tl5-12s in CHO-K1 cells

Glycosylation of l8tl5-l2s in CHO-K1 cells was confirmed using the Protein Deglycosylation Mix II kit (New England Biolabs), according to the manufacturer’s instructions. Figure 32 shows an example SDS PAGE of deglycosylated and non- deglycosylated l8tl5-l2s. Deglycosylation reduces the molecular weight of l8tl5-l2s as seen in Figure 32, lane 4.

Example 33: Recombinant protein quantitation of 18tl5-12s complexes

The l8tl5-l2s complex was detected and quantified using standard sandwich ELISA methods (see Figures 33-36). Anti-human tissue factor antibody served as the capture antibody and biotinylated anti-human IL-12, IL-15, or IL-18 antibody (BAF 219, BAM 247, D045-6, all R&D Systems) served as the detection antibody. Tissue factor in purified 18tl 5-l2s protein complexes was also detected using an anti-human tissue factor capture antibody (143), and anti-human tissue factor antibody detection antibody. The I43/anti-TF Ab ELISA was compared to purified tissue factor at similar concentrations.

Example 34: Immunostimulatory capacity of the 18tl5-12s complex

To assess the IL-15 immunostimulatory activity of the 18tl 5-l2s complex, increasing concentrations of l8tl5-l2s was added to 32ϋb cells (104 cell/well) in 200 pL IMDM: 10% FBS media. The 32ϋb cells were incubated for 3 days at 37°C. On the fourth day, WST-l proliferation reagent (10 pL/well) was added and after 4 hours, absorbance was measured at 450 nm to determine cell proliferation based on cleavage of WST-l to a soluble formazan dye. Bioactivity of human recombinant IL-15 was assessed as a positive control. As shown in Figure 37, l8tl5-l2s demonstrated IL- 15 -dependent cell proliferation of 32ϋb cells. The 18tl 5-l2s complex demonstrated reduced activity compared to human recombinant IL-15, possibly due to the linkage of IL-18 and tissue factor to the IL-15 domain.

In order to assess the individual activities of IL-12 and IL-18 in the l8tl5-l2s complex, l8tl5-l2s was added to HEK-Blue IL-12 and HEK-Blue IL-18 reporter cells (5xl0 ⁴ cell/well; hkb-il 12 and hkb-hmill8, InvivoGen) in 200 pL IMDM: l0% heat- inactivated FBS media. Cells were incubated for overnight at 37°C. 20 mΐ of induced HEK-Blue IL-12 and HEK-Blue IL-18 reporter cell supernatant was added to 180 mΐ of QETANTI-Blue (InvivoGen), and incubated for 1-3 hours at 37°C. IL-12 or IL-18 activity was assessed by measuring absorbance at 620 nm. Human recombinant IL-12 or IL-18 was assessed as a positive or negative control. As shown in Figures 38 and 39, the each of the cytokine domains of the l8tl5-l2s complex retain specific biological activity. The activity of 18tl 5-l2s was reduced compared to that of human recombinant IL-18 or IL- 12, possibly due to linkage of IL-15 and tissue factor to the IL-18 domain and linkage of

IL-12 to the IL-l5Ra sushi domain.

Example 35: Induction of cytokine-induced memory-like NK cells by the 18tl5-12s complex

Cytokine-induced memory-like NK cells can be induced ex vivo following overnight stimulation of purified NK cells with saturating amounts of IL-12 (10 ng/mL), IL-15 (50 ng/mL), and IL-18 (50 ng/mL). These memory -like properties have been measured through expression of IL-2 receptor a (IL-2Ra, CD25), CD69 (and other activation markers), and increased IFN-g production. To evaluate the ability of l8tl5-l2s complexes to promote generation of cytokine-induced memory-like NK cells, purified human NK cells (>95% CD56+) were stimulated for 14-18 hours with O.OlnM to lOOOOnM of the 18tl 5-l2s complex or a combination of individual cytokines

(recombinant IL-12 (10 ng/mL), IL-18 (50 ng/mL), and IL-15 (50 ng/mL)). Cell-surface CD25 and CD 69 expression and intracellular IFN-g levels were assessed by antibody- staining and flow cytometry. Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells were isolated with the Rosette Sep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16- BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend). Cells were counted and resuspended in 0.2 x l0 ⁶ /mL in a 96 well flat bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco), supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). Cells were stimulated with either a mixture of cytokines hIL-l2 (10 ng/mL) (Biolegend), hIL-l8 (50 ng/mL) (R&D Systems) and hlL- 15 (50 ng/mL) (NCI) or with 0.01 nM to lOOOOnM of the l8tl5-l2s at 37°C, 5% C0 ₂ for

14-18 hrs. The cells were then harvested and surface stained with CD56-BV421, CD16- BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend) for 30 minutes. After staining, cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (IX PBS (Hyclone), with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes, cells were analyzed using a BD FACSCelesta™ flow cytometer (Plotted Data-Mean Fluorescence Intensity, see Figures 40A and 40B).

Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells were isolated with the Rosette Sep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16- BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend). Cells were counted and resuspended in 0.2 x l0 ⁶ /mL in a 96 well flat bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco), supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). Cells were stimulated with either a cytokine mix of hIL-l2 (10 ng/mL) (Biolegend), l 8 (50 ng/mL) (R&D), and hIL- 15 (50

ng/mL) (NCI), or 0.01 nM to 10000 nM of the 18tl 5- l2s complex at 37°C, 5% C02 for 14-18 hrs. The cells were then treated with 10 pg/mL of Brefeldin A (Sigma) and IX of Monensin (eBioscience) for 4 hrs before harvesting and staining for CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 for 30 minutes. After staining, cells were washed (1500 RPM for 5 minutes in room temperature) in FACS buffer (IX PBS

(Hyclone), with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) and fixed for 10 minutes at room temperature. After fixation, cells were washed (1500 RPM for 5 minutes in room temperature) in lx permeabilized buffer (eBioscience) and stained with IFN-g- PE Ab (Biolegend) for 30 minutes at room temperature. Cells were washed once again with lx permeabilized buffer and then washed with FACS buffer. Cell pellets were resuspended in 300 pls of FACS buffer and analyzed using a BD FACSCelesta™ flow cytometer (Plotted % of IFN-g Positive Cells, see Figure 41).

Example 36: In vitro cytotoxicity of NK cells against human tumor cells

Human myelogenous leukemia cells, K562 (CellTrace violet labelled), were incubated with purified human NK cells in the presence of increasing concentrations of the 18tl 5-l2s complex or a mixture of cytokines as a control. After 20 hours, the cultures were harvested, stained with propidium iodide (PI), and assessed by flow cytometry. As shown in Figure 42, the l8tl5-l2s complex induced human NK cytotoxicity against K562, at levels similar or greater than the cytokine mixture, wherein both the 18tl 5-l2s complex and the cytokine mixture induced greater cytotoxicity than the medium control.

Example 37: Creation of IL-12/IL-15RaSu/aCD16scFv and IL-18/TF/IL-15 DNA constructs

In a non-limiting example, IL-l2/IL-l5RaSu/aCDl6scFv and IL-18/TF/IL-15 DNA constructs were created (see Figure 43). The human IL-12 subunit sequences, human IL-l5RaSu sequence, human IL-15 sequence, human tissue factor 219 sequence, and human IL-18 sequence were synthesized by Genewiz. A DNA construct was made linking the IL-12 subunit beta (p40) to IL-12 subunit alpha (p35) with a GS (3) linker to generate a single chain version of IL-12, directly linking the IL-12 sequence to the IL- l5RaSu sequence, and directly linking the IL-12/ IL-l5RaSu construct to the N-terminus coding region of aCDl6scFv (referred as l8tl5-l2sl6).

The nucleic acid sequence of the IL-l2/IL-l5RaSu/aCDl6scFv construct is as follows (SEQ ID NO: 156): (Signal peptide)

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC

(Human IL-12 subunit beta (p40))

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTAT CCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAG ACGGC AT C ACTTGGACCCTCGAT C AGAGC AGCGAGGT GCTGGGCTCCGGAAA GACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGC CACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGG AAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGA ATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGT TGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCG GGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCT GAGAGGGTTCGT GGCGAT AAC AAGGAAT ACGAGT AC AGCGTGGAGT GCC AA GAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGG TGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATC CGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCA A A A AT AGCC GGC A AGTTGAGGT C TC TT GGGA AT ATCCC GAC AC TT GGAGC AC ACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCA AGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCA TCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCC AGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC

{Linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

{Human IL-12 subunit alpha (p35))

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTAC

ACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAG

GCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGAT

ATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGA

CAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGG

CTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTC

CATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCC AAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGG

CTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCT

CAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGT

GCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATG

AGCTATTTAAACGCCAGC

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

(anti-Human CD16 light chain variable domain)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACC

GTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGT

ACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAA

CAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCG

CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTG

CAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAG

CTGACCGTGGGCCAT

{Linker)

GGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCC

(anti-Human CD16 heavy chain variable domain)

G AGGT GC AGC T GGT GG AGT C C GG AGG AGG AGT GGT G AGGC C T GG AGG CTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCA TGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCAT CAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTC ACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCC TGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCT GTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG

Constructs were also made linking the IL-18 sequence to the N-terminus coding region of tissue factor 219, and linking the IL-18/TF construct with the N-terminus coding region of IL-15 (see Figure 44). The nucleic acid sequence of the IL-18/TF/IL-15 construct (including leader sequence) is as follows (SEQ ID NO: 1 19):

(Signal peptide)

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCT

ACAGC

(Human IL-18)

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAC GACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGAC CGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGT ACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGA GAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATG AACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGC GGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGA GGGC T AC TTTTT AGC TT GT GA A A AGGAGAGGGATTT ATT C A AGC T GATCC T C A AGAAGGAGGACGAGCTGGGCGATCGTTCC AT CAT GTT C ACCGTCC AA AACGA GGAT

(Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Human IL-15) AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

Example 38: Secretion of IL-12/IL-15RaSu/aCD16scFv and IL-18/TF/IL-15 fusion proteins The IL-l2/IL-l5RaSu/aCDl6scFv and IL-18/TF/IL-15 constructs were cloned into a pMSGV-l modified retrovirus expression vector (Hughes, Hum Gene Ther 16:457-72, 2005, herein incorporated by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells resulted in secretion of a soluble IL-l8/TF/IL-l5:IL-l2/IL-l5RaSu/aCDl6scFv protein complex (referred to as l8tl5-l2s/aCDl6). Co-expression of the two constructs in CHO- Kl cells resulted in secretion of the soluble IL-18/TF/IL-15TL-12/IL- l5RaSu/aCDl6scFv protein complex (referred to as l8tl5-l2s/aCDl6, see Figures 45 and 46), which can be purified by anti-TF antibody affinity and other chromatography methods. In some cases, the signal peptide is cleaved from the intact polypeptide to generate the mature form.

The amino acid sequence of the IL-l2/IL-l5RaSu/aCDl6scFv fusion protein (including signal peptide sequence) is as follows (SEQ ID NO: 155):

(Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-12 subunit beta (p40))

IWELKKD VYVVELDWYPD APGEMVVLT CDTPEEDGITWTLDQ S SEVLGS GKTLTIQ VKEF GD AGQ YTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKN KTFLRCEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQGVTCGAATLS AERV RGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPD PPKNLQLKPLKN SRQVEV SWEYPDTW STPHS YF SLTF C VQ VQGKSKREKKDRVF TDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

{Linker)

GGGGS GGGGS GGGGS

{Human IL-12 subunit alpha (p35))

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHE DITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYE DLKMY QVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQ ALNFN SETVPQKS S LEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNKAT NVAHWTTPSLKCIR

(anti-Human CD16 light chain variable domain)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRP S GIPDRF S GS S S GNT ASLTIT GAQ AEDE AD Y Y CN SRD S S GNH V VF GGGTKL TVGH

{Linker)

GGGGS GGGGS GGGGS

(anti-Human CD16 heavy chain variable domain)

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWV SGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRS LLFD YW GQGTL VT V SR

The amino acid sequence of the IL-18/TF/IL-15 fusion protein (including leader sequence) is as follows (SEQ ID NO: 1 18):

(Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-18)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISM

YKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPG

HDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS

Example 39: Creation of IL-18/IL-15RaSu and IL-12/TF/IL-15 DNA constructs

In a non-limiting example, IL-l8/IL-l5RaSu and IL-12/TF/IL-15 DNA constmcts were created. The human IL-18 subunit sequences, human IL-l5RaSu sequence, human IL-12 sequence, human tissue factor 219 sequence, and human IL-15 sequence were synthesized by Genewiz. A DNA construct was made linking IL-18 directly to IL- l5RaSu. An additional construct was also made linking IL-12 sequence to the N- terminus coding region of human tissue factor 219 form, and further linking the IL-12/TF construct to the N-terminus coding region of IL-15. As described above, a single-chain version of IL-12 (p40-linker-p35) was used.

The nucleic acid sequence of the IL-l8/IL-l5RaSu construct (including signal peptide sequence) is as follows (SEQ ID NO: 189):

(Signal peptide)

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCT

ACAGC

(Human IL-18)

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAC GACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGAC CGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGT ACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGA GAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATG AACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGC GGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGA GGGC T AC TTTTT AGC TT GT GA A A AGGAGAGGGATTT ATT C A AGC T GATCC T C A AGAAGGAGGACGAGCTGGGCGATCGTTCC AT CAT GTT C ACCGTCC AA AACGA GGAT

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG

AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA

AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA

CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The nucleic acid sequence of the IL-12/TF/IL-15 construct (including leader sequence) is as follows (SEQ ID NO: 190):

(Signal peptide)

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCT

ACTCC

(Human IL-12 subunit beta (p40))

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTAT CCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAG ACGGC AT C ACTTGGACCCTCGAT C AGAGC AGCGAGGT GCTGGGCTCCGGAAA GACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGC CACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGG AAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGA ATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGT TGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCG GGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCT GAGAGGGTTCGT GGCGAT AAC AAGGAAT ACGAGT AC AGCGTGGAGT GCC AA GAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGG TGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATC CGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCA A A A AT AGCC GGC A AGTTGAGGT C TC TT GGGA AT ATCCC GAC AC TT GGAGC AC ACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCA

AGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCA

TCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCC

AGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC

{Linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

{Human IL-12 subunit alpha (p35))

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTAC

ACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAG

GCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGAT

ATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGA

CAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGG

CTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTC

CATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCC

AAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGG

CTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCT

CAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGT

GCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATG

AGCTATTTAAACGCCAGC

(Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG

CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT

TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT

ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA

ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC

ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA

CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC

ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC

AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT

GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA

AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

Example 40: Secretion of IL-18/IL-15RaSu and IL-12/TF/IL-15 fusion proteins

The IL-l8/IL-l5RaSu and IL-12/TF/IL-15 constructs were cloned into a pMSGV- 1 modified retrovirus expression vector (Hughes, Hum Gene Hier 16:457-72, 2005 herein incorporated by reference), and the expression vector was transfected into CHO- Kl cells. Co-expression of the two constructs in CHO-K1 cells resulted in secretion of a soluble IL-l2/TF/IL-l5:IL-l8/IL-l5RaSu protein complex (referred to as l2tl 5/sl 8), which can be purified by anti-TF antibody affinity and other chromatography methods.

The amino acid sequence of the IL-l8/IL-l5RaSu fusion protein (including signal peptide sequence) is as follows (SEQ ID NO: 191):

(Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-18)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISM YKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPG HDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNKAT NVAHWTTPSLKCIR

The amino acid sequence of the IL-12/TF/IL-15 fusion protein (including leader sequence) is as follows (SEQ ID NO: 192): (Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-12 subunit beta (p40))

IWELKKD VYVVELDWYPD APGEMVVLT CDTPEEDGITWTLDQ S SEVLGS GKTLTIQ VKEF GD AGQ YTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKN KTFLRCEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQGVTCGAATLS AERV RGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPD PPKNLQLKPLKN SRQVEV SWEYPDTW STPHS YF SLTF C VQ VQGKSKREKKDRVF TDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

{Linker)

GGGGS GGGGS GGGGS

{Human IL-12 subunit alpha (p35))

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHE DITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYE DLKMY QVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQ ALNFN SETVPQKS S LEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted. Example 41: Recombinant protein quantitation of the 18tl5-12sl6 complex

The l8tl5-l2sl6 complex (comprising IL-l2/IL-l5RaSu/aCDl6scFv;IL-l8/TF/IL-l5) was detected and quantified using standard sandwich ELISA methods (see Figure 47). Anti-human tissue factor antibody /IL-2 or anti-TF antibody/IL-l8 served as the capture antibody and biotinylated anti-human IL-12 or IL-18 antibody (BAF 219, D045-6, both R&D Systems) served as the detection antibody. Tissue factor was also detected using an anti-human tissue factor antibody (143), and anti-human tissue factor antibody detection antibody.

Example 42: Creation of TGF RII/IL-15RaSu and IL-21/TF/IL-15 DNA constructs In a non-limiting example, a TGF RII/IL- 15RaSu DNA construct was created

(see Figure 48). The human TGF RII dimer and human IL-21 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the TGF RII to another TGF RII with a linker to generate a single chain version of TGF RII and then directly linking the TGF RII single chain dimer sequence to the N-terminal coding region of IL- 15RaSu.

The nucleic acid sequences of the TGF RII/IL- 15RaSu construct (including signal sequence) is as follows (SEQ ID NO: 133):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC

(Human TGFpRII- 1 ^st fragment)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT {Linker)

GGAGGT GGCGGATCCGGAGGT GGAGGTTCTGGTGGAGGT GGGAGT

(Human TGF PRII-2 ^nd fragment)

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACC

GATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGA

GGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCAC

CTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAAT

GACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACC

ACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAA

AAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGC

AACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(Human IL-15R a sushi domain)

ATCACGTGTCCTCCTCCTATGTCCGTGGAACACGCAGACATCTGGGTC AAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAA GCGT AAAGCCGGC ACGTCC AGCCTGACGGAGTGCGT GTT GAAC AAGGCC ACG AATGTCGCCCACTGGACAACCCCCAGTCTCAAATGTATTAGA

Additionally, an IL-21/TF/IL-15 construct was made linking the IL-21 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-21/TF construct to the N-terminus coding region of IL-15 (see Figure 49). The nucleic acid sequence of the IL-21/TF/IL-15 construct (including leader sequence) is as follows (SEQ ID NO: 129):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT

CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT

GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC

AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA

ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCC AAGGAGTTCCTGGAGAGGTTC AAGTCCCTGCTGC AGAAGAT GAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(Human Tissue Factor 219)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGC ACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTT ACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTA CACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAG CAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCAC CGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATT TAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCAC C A AGGT G A AC GT C AC C GT C G AGG AT G A A AGG AC TT T AGT GC GGC GG A AT A AC ACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTA CTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAAC GAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAG CCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGA GTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

Example 43: Secretion of TGFpRII/IL-15RaSu and IL-21/TF/IL-15 fusion proteins

The TGF RII/IL- 1 5RaSu and IL-21/TF/IL-15 DNA constructs were cloned into a pMSGV-l modified retrovirus expression vector (as described in Hughes et al., Hum Gene Ther 16:457-72, 2005, herein incorporated by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells resulted in secretion of the soluble IL-21 /TF/IL- 15 :TGFPRII/IL- 15RaSu protein complex (referred to as 2ltl5-TGFRs, see Figures 50 and 51). The 2ltl5-TGFRs complex was purified from CHO-K1 cell culture supernatant using anti-TF antibody affinity chromatography and other chromatography methods.

The amino acid sequence of the TGF RII/IL-l5RaSu construct (including signal peptide sequence) is as follows (SEQ ID NO: 132):

(Signal peptide)

MKWVTFISLLFLF S SAYS

(Human TGFpRII- 1 ^st fragment)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI

TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMC S C S SDECNDNIIF SEEYNT SNPD

{Linker)

GGGGS GGGGS GGGGS

(Human TGF bKII-2 ^hά fragment)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMC S C S SDECNDNIIF SEEYNT SNPD

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNKAT

NVAHWTTPSLKCIR

The amino acid sequence of the mature IL-21/TF/IL-15 fusion protein (including signal peptide sequence) is as follows (SEQ ID NO: 128):

(Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLP APEDVETNCEW S AF SCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFK SLLQKMIHQHL S SRTHGSED S

(Human Tissue Factor 219) S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS In some cases, the leader peptide is cleaved from the intact polypeptide to generate a mature form that may be soluble or secreted.

Example 44: Purification of 21tl5-TGFRs by immunoaffinity chromatography

An anti-TF antibody affinity column was connected to a GE Healthcare AKTA™ Avant protein purification system. The flow rate was 4 mL/min for all steps except the elution step, which was 2 mL/min.

Cell culture harvest of 2ltl5-TGFRs was adjusted to pH 7.4 with 1M Tris base and loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes PBS, followed by elution with 6 column volumes 0.1M acetic acid, pH 2.9. Absorbance at 280 nm was collected and then the sample was then neutralized to pH 7.5-8.0 by adding 1M Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon® centrifugal filters with a 30 KDa molecular weight cutoff. Figure 52 shows that the 2ltl5-TGFRs complex binds anti-TF antibody affinity column, wherein TF is a 2 ltl 5- TGFRs binding partner. The buffer-exchanged protein sample is stored at 2-8°C for further biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was then stripped using 6 column volumes 0.1M glycine, pH 2.5. The column was then neutralized using 10 column volumes PBS, 0.05% sodium azide, and stored at 2-8°C. Example 45: Size exclusion chromatography of 21tl5-TGFRs

A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration column was connected to a GE Healthcare AKTA™ Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.8 mL/min. A capillary loop was used to inject 200pL of 1 mg/mL of 2ltl5-TGFRs complex onto the column. The injection was then chased with 1.25 column volumes of PBS. The SEC chromatograph was shown in Figure 53. There were two protein peaks, likely representing a monomer and dimer forms of 2ltl5-TGFRs.

Example 46: SDS-PAGE of 21tl5-TGFRs To determine the purity and protein molecular weight, the purified 2ltl5-TGFRs complex protein sample was analyzed using 4-12% NuPage Bis-Tris protein gel SDS- PAGE under reduced conditions. The gel was stained with InstantBlue™ for about 30 min, followed by destaining overnight in purified water. Figure 54 shows an example SDS gel of anti-TF antibody affinity purified 2ltl5-TGFRs, with bands at 39.08 kDa and 53 kDa. Glycosylation of 2ltl5-TGFRs in CHO cells was confirmed using the Protein

Deglycosylation Mix II kit (New England Biolabs) and the manufacturer’s instructions. Figure 10 shows an example SDS PAGE of deglycosylated and non-deglycosylated 2ltl5-TGFRs. Deglycosylation reduces the molecular weight of 2ltl5-TGFRs, as seen in lane 4 of Figure 54. Example 47: Recombinant protein quantitation of 21tl5-TGFRs complexes

The 2ltl5-TGFRs complex was detected and quantified using standard sandwich ELISA methods (see Figures 55-59). Anti-human tissue factor antibody served as the capture antibody and biotinylated anti-human IL-21, IL-15, or TGF RII served as the detection antibody. Tissue factor was also detected using an anti-human tissue factor capture antibody (143), and anti-human tissue factor antibody detection antibody. The I43/anti-TF Ab ELISA was compared to purified tissue factor at similar concentrations. Example 48: Immunostimulatory capacity of the 21tl5-TGFRs complex

To assess the IL-15 immunostimulatory activity of the 2ltl5-TGFRs complexes, increasing concentrations of 2ltl5-TGFRs was added to 32ϋb cells (10 ⁴ cell/well) in 200 pL IMDM: 10% FBS media and cells were incubated for 3 days at 37°C. On the fourth day, WST-l proliferation reagent (10 pL/well) then was added and after 4 hours, absorbance was measured at 450 nm to determine cell proliferation based on cleavage of WST-l to a soluble formazan dye. Bioactivity of the human recombinant IL-15 was assessed as a positive control. As shown in Figure 59, 2ltl5-TGFRs demonstrated IL- 15-dependent 32ϋb cell proliferation. The 2ltl5-TGFRs complex was reduced compared to that of human recombinant IL-15, possibly due to the linkage of IL-21 and tissue factor to the IL-15 domain.

Additionally, HEK-Blue TGF reporter cells (hkb-tgfb, InvivoGen) were used to measure the ability of 2ltl5-TGFRs to block THRbI activity (Figure 60).

Increasing concentrations of 2ltl5-TGFRs were mixed with 0.1 nM of TGF i and added to HEK-Blue THRb reporter cells (2.5xl0 ⁴ cell/well) in 200 pL

IMDM: 10% heat-inactivated FBS media. Cells were incubated overnight at

37°C. The next day, 20 pl of induced HEK-Blue TGF reporter cell supernatant was added to 180 pl of QETANTI-Blue (InvivoGen) and incubated for 1-3 hours at 37°C. 2ltl5-TGFRs activity was assessed by measuring absorbance at 620 nm.

Human recombinant TGF RII/Fc activity was assessed as a positive control.

These results demonstrate that THRbKII domain of the 2ltl5-TGFRs

complex retains its ability to trap THRbI. The ability of 2ltl5-TGFRs to block

THRbI activity was reduced compared to that of human recombinant

TORbIHI/Ro, possibly due to the linkage of TGF RII to the IL-l5Ra sushi

domain.

Example 49: Induction of cytokine-induced memory-like NK cells by the 21tl5- TGFRs complex

Cytokine-induced memory-like NK cells can be induced ex vivo following overnight stimulation of purified NK cells with saturating amounts of cytokines. These memory -like properties can be measured through expression of IL-2 receptor a (IL-2Ra, CD25), CD69 (and other activation markers), and increased IFN-g production. To evaluate the ability of 2ltl5-TGFRs complexes to promote generation of cytokine- induced memory-like NK cells, purified human NK cells (>95% CD56+) were stimulated for 14-18 hours with 1 nM to 100 nM of the 2ltl5-TGFRs complex. Cell-surface CD25 and CD 69 expression and intracellular IFN-g levels were assessed by antibody-staining and flow cytometry.

Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells were isolated with the Rosette Sep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16- BV510, CD25-PE, CD69-APCFire750 (BioLegend). Cells were counted and

resuspended in 0.2 x l0 ⁶ /mL in a 96 well flat bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco), supplemented with 2 mM L-glutamine (Thermo Life

Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). Cells were stimulated with either mix- cytokines of ML-21 (50 ng/mL) (Biolegend) and hH.- l 5 (50 ng/mL) (NCI) or with 1 nM, 10 nM, or 100 nM 2ltl5-TGFRs complex overnight at 37°C, 5% CO2 for 14-18 hrs. The cells were then harvested and surface stained for CD56-BV421, CD16-BV510, CD25- PE, CD69-APCFire750 for 30 minutes. After staining, cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (IX PBS (Hyclone) with 0.5% BSA

(EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes, cells were analyzed using a BD FACSCelesta™ flow cytometer (Plotted Data-Mean Fluorescence Intensity, see Figures 61 A and 61B).

Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells were isolated with the Rosette Sep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16- BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend). Cells were counted and resuspended in 0.2 x l0 ⁶ /mL in a 96 well flat bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco), supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). Cells were stimulated with either mix- cytokines of ML-21 (50 ng/mL) (Biolegend) and hIL- 15 (50 ng/mL) (NCI) or with 1 nM, 10 nM, or 100 nM 2ltl5-TGFRs complex overnight at 37°C, 5% C02 for 14-18 hrs. The cells were then treated with 10 pg/mL of Brefeldin A (Sigma) and IX of Monensin (eBioscience) for 4 hrs. Cells were harvested and surface stained with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies for 30 minutes. After staining, cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (IX PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) and fixed for 10 minutes at room temperature. After fixation, cells were washed (1500 RPM for 5 minutes at room temperature) with lx permeabilized buffer

(eBioscience) and stained for intracellular IFN-g- PE Ab (Biolegend) for 30 minutes at room temperature. Cells were washed once again with lx permeabilized buffer and then washed with FACS buffer. Cell pellets were resuspended in 300 pls of FACS Buffer and analyzed using a BD FACSCelesta™ flow cytometer. (Plotted % of IFN-g Positive Cells, see Figure 62). Example 50: In vitro cytotoxicity of NK cells against human tumor cells

K562 (CellTrace violet labelled), human myelogenous leukemia cells, were incubated with purified human NK cells (using StemCell human NK cell purification kit (E:T ratio; 2: 1)) in the presence of increasing concentrations of the 2ltl5-TGFRs complex. After 20 hours, the cultures were harvested, stained with propidium iodide (PI), and assessed by flow cytometry. As shown in Figure 63, the 2ltl5-TGFRs complex induced human NK cytotoxicity against K562, as compared to control.

Example 51: Creation of an IL-21/TF mutant/IL-15 DNA construct and resulting fusion protein complex with TGF RII /IL-15RaSu

In a non-limiting example, an IL-21/TF mutant/IL-l5 DNA construct was made by linking IL-21 directly to the N-terminus coding region of a tissue factor 219 mutant, and further linking the IL-21/TF mutant to the N-terminus coding region of IL-15.

The nucleic acid sequence of the IL-21/TF mutant/IL-l5 construct (including signal peptide sequence) is as follows (SEQ ID NO: 193, shaded nucleotides are mutant and the mutant codons are underlined): (Signal sequence)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCC A AGGAGTTCCTGGAGAGGTTC AAGTCCCTGCTGC AGAAGAT GAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(Human Tissue Factor 219 mutants)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGC ACCAACTTCGCGACAGCTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTT ACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTA CACAACAGACACCGAGTGTGCTTTAACCGACGAAATCGTCAAGGACGTCAAG CAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCAC CGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATT TAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCAC CAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGGCGCGGAATAA CACAGCTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGT ACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAA CGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAA GCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTG AGT GC AT GGGCC AAGAAAAGGGCGAGTTCCGGGAG

(Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of the IL-21/TF mutant/IL-l5 construct (including signal peptide sequence) is as follows (SEQ ID NO: 194, substituted residues are shaded):

(Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLP APEDVETNCEW S AF SCF

QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFK SLLQKMIHQHL S SRTHGSED S

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNF AT ALEWEPKP VN Q V YT V QI S TK S GD WK SKC FYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S

LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS

In some cases, the leader peptide is cleaved from the intact polypeptide to generate a mature form that may be soluble or secreted.

In some embodiments, the IL-21/TF mutant/IL-l5 DNA construct may be combined with an TGF RII /IL-l5RaSu DNA construct, transfected into cells using a retroviral vector as described above, and expressed as IL-21/TF mutant/IL-l5 and TGF RII/IL- l 5RaSu fusion proteins. The IL-l5RaSu domain of the TGF RII/IL- l5RaSu fusion protein binds to the IL-15 domain of the IL-21/TF mutant/IL-l5 fusion protein to create an IL-21/TF mutant/IL- 15:TGFpRII /IL-l5RaSu complex.

Example 52: Creation of IL-21/IL-15RaSu and TGFpRII/TF/IL-15 DNA constructs and the resulting fusion protein complex In a non-limiting example, an IL-2l/IL-l5RaSu DNA construct was made by linking IL-21 directly to the IL-l5RaSu subunit sequence. The nucleic acid sequence of the IL-2l/IL-l5RaSu construct (including signal sequence) is as follows (SEQ ID NO: 148):

(Signal sequence)

ATGAAGTGGGTGACCTTC ATC AGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCC AAGGAGTTCCTGGAGAGGTTC AAGTCCCTGCTGC AGAAGAT GAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence of the IL-2l/IL-l5RaSu construct (including signal peptide sequence) is as follows (SEQ ID NO: 147):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEW S AF SCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFK SLLQKMIHQHL S SRTHGSED S

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNKAT NVAHWTTPSLKCIR

In some cases, the leader peptide is cleaved from the intact polypeptide to generate a mature form that may be soluble or secreted.

In some embodiments, the IL-2l/IL-l5RaSu DNA construct may be combined with a TGFpRII/TF/IL- l 5 DNA construct, transfected into a retroviral vector as described above, and expressed as IL-2l/IL-l5RaSu and TGFpRII/TF/IL- 15 fusion proteins. The IL-l5RaSu domain of the IL-2l/IL-l5RaSu fusion protein binds to the IL- 15 domain of the TGFpRII/TF/IL- 15 fusion protein to create a TGFpRII/TF/IL- 15:IL-

2l/IL-l5RaSu complex.

The TGF RII/TF/IL- 15RaSu DNA construct was created by linking the TGFpRII sequence to the N-terminus coding region of human tissue factor 219 form, and then linking the TGF RII/TF construct to the N-terminus coding region of IL-15. As described above, a single-chain version of TGF RII (TGF RII-linker-TGF RII) was used. The nucleic acid sequence of the TGF RII/TF/IL-l5 construct (including leader sequence) is as follows (SEQ ID NO: 164):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC

(Human TGFpRII- 1 ^st fragment)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT

GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA

GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT

AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT

{Linker)

GGAGGT GGCGGATCCGGAGGT GGAGGTTCTGGTGGAGGT GGGAGT

(Human TGF PRII-2 ^nd fragment)

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACC GATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGA GGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCAC CTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAAT GACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACC ACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAA AAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGC AACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(Human Tissue Factor 219)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGC ACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTT ACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTA CACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAG CAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCAC CGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATT TAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCAC C A AGGT G A AC GT C AC C GT C G AGG AT G A A AGG AC TT T AGT GC GGC GG A AT A AC ACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTA CTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAAC GAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAG CCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGA GTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of the TGF RII/TF/IL- 1 5 fusion protein (including signal peptide) is as follows (SEQ ID NO: 163):

(Signal peptide)

MKWVTFISLLFLF S SAYS

(Human TGFpRII- 1 ^st fragment)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMC S C S SDECNDNIIF SEEYNT SNPD

Linker )

GGGGS GGGGS GGGGS

(Human TGF RII-2" ^d fragment)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMC S C S SDECNDNIIF SEEYNT SNPD

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI SLE SG

DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN

TS Example 53. Production of an Exemplary Single-Chain Chimeric Polypeptides

An exemplary single-chain chimeric polypeptide including a first target-binding domain that is an anti-CD3 scFv, a soluble human tissue factor domain, and a second target-binding domain that is an anti-CD28 scFv was generated

(aCD3scFv/TF/aCD28scFv) (Figure 64). The nucleic acid and amino acid sequences of this single-chain chimeric polypeptide are shown below.

Nucleic Acid Encoding Exemplary Single-Chain Chimeric Polypeptide

(aCD3scFv/TF/ocCD28scFv) (SEQ ID NO: 103)

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTTATTCAGCTCCGCCT

ATTCC

( aCD3 light chain variable region)

CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGT

GAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACT

GGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCA

GCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCAC

CAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACT

ATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCT

CGAAATCAATCGT

{Linker)

GGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGC

( aCD3 heavy chain variable region)

CAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGC CTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAA T GC ATTGGGT C AAGC AGAGGCCCGGTC AAGGTTT AGAGT GGATCGGAT AT AT CAACCCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCC ACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTT AACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCAC TACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGC (Human tissue factor 219 form)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGC ACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTT ACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTA CACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAG CAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCAC CGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATT TAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCAC C A AGGT G A AC GT C AC C GT C G AGG AT G A A AGG AC TT T AGT GC GGC GG A AT A AC ACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTA CTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAAC GAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAG CCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGA GTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

( aCD28 light chain variable region)

GTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCC GTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCA GT GGGTC AAAC AGA AGCCCGGAC AAGGTCTCGAGT GGATCGGC AGC ATC AA CCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACT TTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAAC ATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTAC T GGGGACGGGGC AC AAC ACTGACCGT GAGC AGC

{Linker)

GG AGGC GG AGGC T C C GGC GG AGGC GG AT C T GGC GGT GGC GGC TC C

( aCD28 light chain variable region)

GACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGC

GGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCAT

TGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCA

GCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAG

CTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTT GTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGAC

AAAGAGG

Exemplary Single-Chain Chimeric Polypeptide (aCD3scFv/TF/aCD28scFv) (SEQ ID NO: 102)

(Signal peptide)

MKWVTFISLLFLF S SAYS

( aCD3 light chain variable region)

QIVLT Q SP AIMS ASPGEK VTMT C S AS S S V S YMNW Y QQKSGT SPKRWIYDT SKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI NR

{Linker)

GGGGS GGGGS GGGGS

( aCD3 heavy chain variable region)

QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWI GYINPSRGYTNYNQKFKDKATLTTDKS S STAYMQLS SLTSEDS AVYYC ARYYDD HYCLD YWGQGTTLT VS S

(Human tissue factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

( aCD28 light chain variable region)

VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGS INPYND YTKYNEKFKGK ATLTSDKS SITAYMEF S SLTSEDS ALYYC ARWGDGNY W GRGTTLT V S S

{Linker)

GGGGS GGGGS GGGGS

( aCD28 heavy chain variable region) DIEMTQ SP AIMS ASLGERVTMTCT AS S S VS S S YFHW YQQKPGS SPKLCIYST SNL A SGVPPRF SGSGST S YSLTIS SME AED AAT YF CHQ YHRSPTF GGGTKLETKR

A second exemplary single-chain chimeric polypeptide including a first target- binding domain that is an anti-CD28 scFv, a soluble human tissue factor domain, and a second target-binding domain that is an anti-CD3 scFv was generated

(aCD28scFv/TF/aCD3scFv) (Figure 65). The nucleic acid and amino acid sequences of this single-chain chimeric polypeptide are shown below.

Nucleic Acid Encoding Exemplary Single-Chain Chimeric Polypeptide

(aCD28scFv/TF/aCD3scFv) (SEQ ID NO: 195)

(Signal peptide)

ATGAAATGGGTCACCTTCATCTCTTTACTGTTTTTATTTAGCAGCGCCT

ACAGC

( aCD28 light chain variable region)

GTGCAGCTGC AGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCC

GTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCA ATGGGTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAAT CCCTACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTC TGACAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACT TCTGAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTG GGGCCGGGGAACTACTTTAACAGTGAGCTCC

(. Linker )

GGCGGCGGCGGAAGCGGAGGT GGAGGATCTGGCGGT GGAGGC AGC

( aCD28 heavy chain variable region)

GACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCTCTTTAGGA

GAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTT CCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTAAACTGTGTATCTACTCCA CAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACC TCTTACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTT TTGCCATCAGTACCACCGGTCCCCTACCTTTGGC GGAGGC ACAAAGCTGGAG ACCAAGCGG (Human tissue factor 219 form)

AGCGGCACCACCAACACAGTGGCCGCCTACAATCTGACTTGGAAATCC ACCAACTTCAAGACCATCCTCGAGTGGGAGCCCAAGCCCGTTAATCAAGTTT ATACCGTGCAGATTTCCACCAAGAGCGGCGACTGGAAATCCAAGTGCTTCTA TACCACAGACACCGAGTGCGATCTCACCGACGAGATCGTCAAAGACGTGAAG CAGACATATTTAGCTAGGGTGTTCTCCTACCCCGCTGGAAACGTGGAGAGCA CCGGATCCGCTGGAGAGCCTTTATACGAGAACTCCCCCGAATTCACCCCCTAT CTGGAAACCAATTTAGGCCAGCCCACCATCCAGAGCTTCGAACAAGTTGGCA C A A AGGT GA AC GT C ACC GT C GA AG AT GAGAGGACTTT AGT GCGGAGGA AC A ATACATTTTTATCCTTACGTGACGTCTTCGGCAAGGATTTAATCTACACACTG TATTACTGGAAGTCTAGCTCCTCCGGCAAGAAGACCGCCAAGACCAATACCA ACGAATTTTTAATTGACGTGGACAAGGGCGAGAACTACTGCTTCTCCGTGCA AGCTGTGATCCCCTCCCGGACAGTGAACCGGAAGTCCACCGACTCCCCCGTG GAGT GC AT GGGCC AAGAGA AGGGAGAGTTTCGT GAG

( aCD3 light chain variable region)

CAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGT GAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACT GGT ATC AGC AGA AGTCC GGC AC C AGC CC T A A A AGGT GGATCT AC GAC AC C AG CAAGCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAACA AGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATT ACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTC GAGATTAATCGT

{Linker)

GG AGGC GG AGGT AGC GG AGG AGGC GG AT C C GGC GGT GG AGGT AGC

( aCD3 heavy chain variable region)

CAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCT

TCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAAT

GCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATC

AACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCA

CCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTT AACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATT

ACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC

Exemplary Single-Chain Chimeric Polypeptide (aCD28scFv/TF/aCD3scFv) (SEQ ID NO: 196)

(Signal peptide)

MKWVTFISLLFLF S SAYS

( aCD28 light chain variable region)

VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGS INPYND YTKYNEKFKGK ATLTSDKS SITAYMEF S SLTSEDS ALYYCARWGDGNY W GRGTTLT V S S

{Linker)

GGGGS GGGGS GGGGS

( aCD28 heavy chain variable region)

DIEMTQ SP AIMS ASLGERVTMTCT AS S S VS S S YFHW YQQKPGS SPKLCIYS TSNL ASGVPPRF SGSGSTS Y SLTIS SMEAED AAT YF CHQYHRSPTF GGGTKLETKR

(Human tissue factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

( aCD3 light chain variable region)

QIVLT Q SP AIMS ASPGEK VTMT C S AS S S V S YMNW Y QQKSGT SPKRWIYDT SKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI NR

{Linker)

GGGGS GGGGS GGGGS

( aCD3 heavy chain variable region) QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWI GYINPSRGYTNYNTQKFKDKATLTTDKS S STAYMQLS SLTSEDS AVYY CARYYDD HYCLD YWGQGTTLT VS S The nucleic acid encoding aCD3scFv/TF/aCD28scFv was cloned into a modified retrovirus expression vectors as described previously (Hughes et al., Hum Gene Ther 16:457-72, 2005). The expression vector encoding aCD3scFv/TF/aCD28scFv was transfected into CHO-K1 cells. Expression of the expression vector in CHO-K1 cells allowed for secretion of the soluble aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide (referred to as 3t28), which can be purified by anti-TF antibody affinity and other chromatography methods.

An anti-tissue factor affinity column was used to purify the

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide. The anti-tissue factor affinity column was connected to a GE Healthcare AKTA Avant system. A flow rate of 4 mL/min was used for all steps except the elution step, which was 2 mL/min.

Cell culture harvest including aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide was adjusted to pH 7.4 with 1M Tris base and loaded onto the anti-TF antibody affinity column (described above) which was equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes PBS, followed by elution with 6 column volumes 0.1 M acetic acid, pH 2.9. An A280 elution peak was collected and then neutralized to pH 7.5-8.0 by adding 1 M Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 kDa molecular weight cutoff. The data in Figure 65 show that the anti tissue factor affinity column can bind the aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide, which contains a human soluble tissue factor domain. The buffer- exchanged protein sample was stored at 2-8 °C for further biochemical analysis and biological activity testing.

After each elution, the anti-tissue factor affinity column was stripped using 6 column volumes of 0.1 M glycine, pH 2.5. The column was then neutralized using 10 column volumes of PBS, 0.05% NaN3, and stored at 2-8 °C. Analytical size exclusion chromatography (SEC) was performed on the aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide using a Superdex 200 Increase 10/300 GL gel filtration column (from GE Healthcare) connected to an AKTA Avant system (from GE Healthcare). The column was equilibrated with 2 column volumes of PBS. A flow rate of 0.8 mL/min was used. Two hundred pL of

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide (1 mg/mL) was injected onto the column using a capillary loop. After injection of the single-chain chimeric polypeptide, 1.25 column volumes of PBS were flowed into the column. The SEC chromatograph is shown in Figure 66. The data show that there are 3 protein peaks, likely representing a monomer and dimer or other different forms of the

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide.

To determine the purity and protein molecular weight of the

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide, the purified

aCD3scFv/TF/aCD28scFv protein sample from anti-tissue factor affinity column was analyzed by standard sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis- Tris gel) electrophoresis (SDS-PAGE) method under reduced conditions. The gel was stained with InstantBlue for about 30 minutes and destained overnight with purified water. Figure 67 shows the SDS gel of the aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide purified using an anti-tissue factor affinity column. The results show that the purified aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide has the expected molecular weight (72 kDa) in reduced SDS gel.

Example 54. Functional Characterization of aCD3scFv/TF/aCD28scFv Single- Chain Chimeric Polypeptide

ELISA-based methods confirmed the formation of the aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide. The aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide was detected using an anti-TF antibody (I43)/anti-TF antibody- specific ELISA with a capture antibody, anti-human tissue factor antibody (143), and a detection antibody, anti-TF antibody (Figure 68). A purified tissue factor protein with a similar concentration was used as a control. A further in vitro experiment was performed to determine whether the

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide is capable of activating human peripheral blood mononuclear cells (PBMCs). Fresh human leukocytes were obtained from the blood bank and peripheral blood mononuclear cells (PBMC) were isolated using density gradient Histopaque (Sigma). The cells were counted and resuspended in 0.2 x l0 ⁶ /mL in a 96-well flat bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were stimulated with aCD3scFv/TF/aCD28scFv single- chain chimeric polypeptide from 0.01 nM to 1000 nM for 3 days at 37 °C, 5% CO2. After 72 hours, the cells were harvested and surface stained for CD4-488, CD8-PerCP Cy5.5, CD25-BV421, CD69-APCFire750, CD62L-PE Cy7, and CD44-PE specific antibodies (Biolegend) for 30 minutes. After surface staining, the cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (IX PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes, the cells were resuspended in 300 pL of FACS buffer and analyzed by Flow Cytometry (Celesta-BD Bioscience). The data in Figures 69 and 70 show that the aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide is able to stimulate both CD8 ⁺ and CD4 ⁺ T-cells.

A further experiment was performed, in which PBMCs isolated from blood using

Histopaque (Sigma) were counted and resuspended in 0.2 x l0 ⁶ /mL in a 96-well flat bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were then stimulated with the aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide from 0.01 nM to 1000 nM for 3 days at 37 °C, 5% CO2. After 72 hours, the cells were harvested and surface stained for CD4-488, CD8-PerCP Cy5.5, CD25-BV421, CD69- APCFire750, CD62L-PE Cy7, and CD44-PE (Biolegend) for 30 minutes. After surface staining, the cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (IX PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes, the cells were resuspended in 300 pL of FACS buffer and analyzed by Flow Cytometry (Celesta-BD Bioscience). The data again show that the aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide was able to stimulate activation of CD4 ⁺ T cells (Figure 71).

Example 55. Construction of exemplary multi-chain chimeric polypeptides and evaluation of properties thereof

Two multi-chain chimeric polypeptides were generated and their properties were evaluated. Each of the two multi-chain chimeric polypeptides included a first chimeric polypeptide that included a soluble tissue factor domain covalently linked a first target- binding domain and a first domain of an affinity pair of domains. The second chimeric polypeptide in each of the two multi-chain chimeric polypeptides included a second domain of the affinity pair of domains, and a second target-binding domain.

Description of logic underlying construction of multi-chain chimeric polypeptides

Tissue Factor (TF) is a stable, transmembrane protein containing 236 amino acid residues. The truncated, recombinant 219-amino-acid extracellular domain of tissue factor is soluble and is known to be expressed at high levels in bacteria or mammalian cells. Without wishing to be bound to a particular theory, the applicants speculated that the 2l9-aa tissue factor could be used as a connector linker for creation of unique multi- chain chimeric polypeptides.

First chimeric polypeptides including soluble tissue factor domain were produced at high levels by CHO-K1 cells grown in fermentation broth. These first chimeric polypeptides were purified by an anti-tissue factor monoclonal antibody (mAh) coupled on a solid matrix. Notably, tissue factor contains binding sites for FVIIa and FX. The catalytic activity of the tissue factor-FVIIa complex for FX is approximately 1 million fold lower when tissue factor is not anchored to a phospholipid bilayer. Thus, without wishing to be bound to a particular theory, applicants speculated that using the 2l9-aa extracellular domain of tissue factor without the transmembrane in construction of the first chimeric polypeptides may eliminate the pro-coagulation activity of tissue factor in the first chimeric polypeptides. In an effort to further reduce or eliminate the pro coagulation activity of the 2l9-aa tissue factor, select mutations in tissue factor can be made, specifically at seven amino acid residues that are known to contribute to binding energy of the FVIIa binding site.

Characterization of binding interactions for described chimeric polypeptides To determine if the first and second chimeric polypeptides bind to each other to form multi-chain chimeric polypeptides, in vitro binding assays were performed. To determine if the first chimeric polypeptide comprising soluble tissue factor domain are recognized and bound by anti-TF mAh, in vitro binding assays were performed. Notably, the data indicated that the mutated tissue factor proteins are still recognized and selectively bound by the anti-TF mAh which is known to bind to the FX binding site on tissue factor. To determine if the first chimeric polypeptides comprising soluble tissue factor domain covalently linked to scFvs or cytokines (see Figure 72 and Figure 73) possess functional scFvs or cytokines, in vitro binding assays were performed. The data from the aforementioned assays were consistent with the purified first chimeric polypeptides having the expected biological activities (e.g., scFvs selectively bind expected target antigens or cytokines selectively bind expected receptors or binding proteins).

In addition, experiments performed using the two multi-chain chimeric polypeptides including a first and second chimeric polypeptide bound to each other demonstrate the expected target binding activity (e.g., the multi-chain chimeric polypeptide binds specifically to the target specifically recognized by the first target- binding domain and the target specifically recognized by the second target-binding domain).

Based on the aforementioned results, applicants concluded that the soluble tissue factor connecter linker provided or enabled appropriate display of the polypeptides encoding either scFvs, interleukins, cytokines, interleukin receptors, or cytokine receptors in three-dimensional space relative to soluble tissue factor domain and relative to one another such that each retained expected biological properties (and activities).

When both the first and second chimeric polypeptides were co-expressed, the heterodimeric complexes were secreted into the fermentation broths at high levels. The complexes were captured and readily purified by anti-TF mAh conjugated to a solid matrix using affinity chromatography. The first and second target-binding domains of these multi-chain chimeric polypeptides retained their expected biological activities as assayed by in vitro binding assays. Thus, the assembly of the multi-chain chimeric polypeptides provides the appropriate spatial display and folding of the domains for biological activities. Importantly, the spatial arrangement of the multi-chain chimeric polypeptides does not interfere with the FX binding site on tissue factor which enables the use of anti-TF mAh for affinity purification.

Characterization of stability for described chimeric polypeptides

Both purified multi-chain chimeric polypeptides are expected to be stable. These multi-chain chimeric polypeptides are structurally intact and expected to be fully biologically active when they are incubated in human serum at 37 °C for 72 hours.

Characterization of propensity of described chimeric polypeptides to aggregate Both purified multi-chain chimeric polypeptides are expected not to form aggregates when stored at 4 °C in PBS.

Characterization of viscosity of described chimeric polypeptides

Compositions including the multi-chain chimeric polypeptides are not expected to have any viscosity issues when formulated at a concentration as high as 50 mg/mL in PBS.

Discussion of multi-chain chimeric polypeptide platform for use in selectively binding clinically relevant targets

The data from our studies show that our platform technologies can be utilized to create molecules that could be fused to target-binding domains derived from antibodies, in any formats as discussed above, adhesion molecules, receptors, cytokines, chemokines etc. With the appropriate target-binding domain, the resulting multi-chain chimeric polypeptides could promote conjugation of various immune effector cells and mediate destruction of target cells, including cancer cells, virally-infected cells, or senescent cells. Other domains in the multi-chain chimeric polypeptides stimulate, activate, and attract the immune system for enhancing cytotoxicity of effector cells for the targeted cells. Example 56: Creation of an IL-7/IL-15RaSu DNA construct

In a non-limiting example, an IL-7/IL-l 5RaSu DNA construct was created (see Figure 72). The human IL-7 sequence, human IL-l 5RaSu sequence, human IL-15 sequence, and human tissue factor 219 sequence were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the IL-7 sequence to the IL-l 5RaSu sequence. The final IL-7/IL-l 5RaSu DNA construct sequence was synthesized by Genewiz.

The nucleic acid sequence encoding the second chimeric polypeptide of IL-7/IL- l 5RaSu construct (including signal peptide sequence) is as follows (SEQ ID NO: 140):

( Signal peptide )

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGG

CC

(Human IL-7)

GATTGT GAT ATT GA AGGT A A AG AT GGC A A AC A AT AT GAGAGT GTTC T A AT GGTC AGC ATCGAT C AATT ATT GGAC AGC AT GAAAGAAATTGGT AGC AATT GCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAG GAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAAT GAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAA CAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCT GGGT GA AGC CCA AC C A AC A A AGAGTTT GGA AGA A A AT A A AT C TTT A A AGGA AC AGA A A A A AC T GA AT GAC TT GT GTTTCC T A A AGAGACT ATT AC A AG AG AT A AAAACTT GTT GGAAT AAAATTTTGAT GGGC ACT AAAGAAC AC

(Human IL-15R a sushi domain)

ATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTC AAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAA GCGT AAAGCCGGC ACGTCC AGCCTGACGGAGTGCGT GTT GAAC AAGGCC ACG AATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGA

The second chimeric polypeptide of IL-7/IL-l 5RaSu construct (including signal peptide sequence) is as follows (SEQ ID NO: 139): (Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A AL GE AQPTK SLEENK SLKEQKKLNDLCFLKRLLQEIKT C WNKILMGTKEH

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGDASIH

DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN

TS

Example 57: Creation of an IL-21/TF/IL-15 DNA construct

In a non-limiting example, an IL-21/TF/IL-15 construct was made (see Figure 73) by linking the IL-21 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-21/TF construct with the N-terminus coding region of IL-15.

The nucleic acid sequence encoding the first chimeric polypeptide of IL- 21/TF/IL-15 construct (including leader sequence), synthesized by Genewiz, is as follows (SEQ ID NO: 129):

(Signal peptide)

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGG

CC

(Human IL-21 fragment)

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATT GTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGC TCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGA

AGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATG

TATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAA

GACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACC

ACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCAT

CAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC

(Human Tissue Factor 219)

T C AGGC ACT AC AAAT ACTGTGGC AGC AT AT AATTT AACTT GGAAAT C A ACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCT ACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTA CACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAA GCAGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGC ACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTA CCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGA ACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAAC AACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACT TT ATT ATTGGAAATCTT C AAGTTC AGGAAAGAAAAC AGCC AAA AC AAAC ACT AATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCA AGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTA GAGT GT AT GGGC C AGGAGA A AGGGGA ATTC AGAGA A

(Human IL-15)

AACTGGGT GAACGT CATC AGC GATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The first chimeric polypeptide of IL-21/TF/IL-15 construct including leader sequence is SEQ ID NO: 128: (Signal peptide)

MGVKVLFALICIAVAEA

(Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEW S AF SCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHL S SRTHGSED S

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKCF YTTD TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQ VGTKVNVTVEDERTLVRRNNTFLSLRDVF GKDLIYTL YYWKS S S SG

KKT AKTNTNEFLID VDKGEN Y CF S VQ A VIP SRT VNRK S TD SP VECMGQEKGEFR

E

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI SLE SG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS

Example 58: Secretion of IL-7/IL-15RaSu and IL-21/TF/IL-15 fusion proteins

The IL-7/IL-l5RaSu and IL-21/TF/IL-15 DNA constmcts were cloned into a pMSGV-l modified retrovirus expression vector (as described by Hughes, Hum Gene Ther 16:457-72, 2005, hereby incorporated by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of a soluble IL-2l/TF/IL-l5:IL-7/IL-l5RaSu protein complex (referred to as 2ltl5-7s, see Figures 74 and 75). The 2ltl5-7s protein was purified from CHO-K1 cell culture supernatant using anti-TF antibody affinity chromatography and size exclusion chromatography resulting in soluble (non-aggregated) protein complexes consisting of IL-7/IL-l5RaSu and IL-21/TF/IL-15 fusion proteins.

In some cases, the leader (signal sequence) peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted. Example 59: Purification of 21tl5-7s by immunoaffinity chromatography

An anti-TF antibody affinity column was connected to a GE Healthcare™ AKTA Avant protein purification system. The flow rate was 4 mL/min for all steps except the elution step, which was 2 mL/min.

Cell culture harvest of 2ltl5-7s was adjusted to pH 7.4 with 1M Tris base and loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes PBS, followed by elution with 6 column volumes 0.1M acetic acid, pH 2.9. Absorbance at 280 nm was collected and then the sample was neutralized to pH 7.5-8.0 by adding 1M Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon® centrifugal filters with a 30 KDa molecular weight cutoff. The buffer-exchanged protein sample was stored at 2-8°C for further biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was then stripped using 6 column volumes 0.1M glycine, pH 2.5. The column was then neutralized using 10 column volumes PBS, 0.05% sodium azide and stored at 2-8 °C.

Example 60: Creation of a TGF- bKEI /IL-15RaSu DNA construct

In a non-limiting example, a TGF-PRII/IL- 15RaSu DNA construct was created (see Figure 76). The human TGF-PRII sequence, human IL-l5RaSu sequence, human IL-15 sequence, and human tissue factor 219 sequence were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the TGF-PRII sequences (separated by a linker) to the IL-l5RaSu sequence. The final TGF-PRII /IL-l5RaSu DNA construct sequence was synthesized by Genewiz. Example 61: Creation of an IL-21/TF/IL-15 DNA construct

In a non-limiting example, an IL-21/TF/IL-15 construct was made (see Figure 77) by linking the IL-21 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-21/TF construct with the N-terminus coding region of IL-15. Example 62: Secretion of TGF- PRII /IL-15RaSu and IL-21/TF/IL-15 fusion proteins

The TGF-PRII /IL-l5RaSu and IL-21/TF/IL-15 DNA constructs were cloned into a pMSGV-l modified retrovirus expression vector (as described by Hughes, Hum Gene Ther 16:457-72, 2005, hereby incorporated by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of a soluble IL-21/TF/IL-15: TGF-PRII/IL- 15RaSu protein complex (referred to as 2ltl5-TGFRs, see Figures 78 and 79). The 2ltl5-TGFRs protein was purified from CHO-K1 cell culture supernatant using anti-TF antibody affinity chromatography and size exclusion chromatography resulting in soluble (non- aggregated) protein complexes consisting of TGF-PRII/IL- 15RaSu and IL-21/TF/IL-15 fusion proteins.

Example 63: Size exclusion chromatography

A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration column was connected to a GE Healthcare AKTA™ Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. A capillary loop was used to inject 200pL of 1 mg/mL of 7tl5-2lscomplex onto the column. The injection was chased with 1.25 column volumes of PBS.

Example 64: SDS-PAGE of 21tl5-7s and 21tl5-TGFRs

To determine the purity and protein molecular weight, the purified 2ltl5-7s or 2ltl5-TGFRs protein sample were analyzed using 4-12% NuPage Bis-Tris protein gel SDS-PAGE. The gel will be stained with InstantBlue™ for about 30 min, followed by destaining overnight in purified water. Example 65: Glycosylation of 21tl5-7s and 21tl5-TGFRs in CHO-K1 cells

Glycosylation of 2 ltl 5-7s in CHO-K1 cells or 2ltl5-TGFRs in CHO-K1 cells were confirmed using the Protein Deglycosylation Mix II kit (New England Biolabs), according to the manufacturer’s instructions. Example 66: Recombinant protein quantitation of 21tl5-7s and 21tl5-TGFRs complexes

The 2ltl5-7s complex or the 2ltl5-TGFRs complex were detected and quantified using standard sandwich ELISA methods. Anti-human tissue factor antibody (IgGl) served as the capture antibody and biotinylated anti-human IL-21, IL-15, or IL-7 antibody (2ltl5-7s) or biotinylated anti-human IL-21, IL-15, or TGF-PRII antibody (2ltl5-TGFRs) served as the detection antibody. Tissue factor in purified 2ltl5-7s or 2ltl5-TGFRs protein complexes was detected using an anti -human tissue factor capture antibody, and anti-human tissue factor antibody (IgGl) detection antibody. The anti-TF ELISA will be compared to purified tissue factor at similar concentrations. Example 67: Creation of an IL-21/IL-15RaSu DNA construct

In a non-limiting example, an IL-2l/IL-l5RaSu DNA construct was created. The human IL-21 sequence and human IL-l5RaSu sequence were obtained from the ETniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the IL-21 sequence to the IL-l5RaSu sequence. The final IL-21/IL- l5RaSu DNA construct sequence was synthesized by Genewiz. See Figure 80.

Example 68: Creation of an IL-7/TF/IL-15 DNA construct

In a non-limiting example, an IL-7/TF/IL-15 construct was made by linking the IL-7 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-7/TF construct with the N-terminus coding region of IL- 15. See Figure 81. Example 69: Creation of an IL-21/IL-15Ra Sushi DNA construct

In a non-limiting example, a second chimeric polypeptide of IL-2l/IL-l5RaSu was generated. The human IL-21 and human IL-l5Ra sushi sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the IL-21 sequence to the IL-l5Ra sushi sequence.

The final IL-2l/IL-l5RaSu DNA construct sequence was synthesized by Genewiz.

The nucleic acid sequence encoding the second chimeric polypeptide of IL-21/IL- l5RaSu domain (including leader sequence), synthesized by Genewiz, is as follows (SEQ ID NO: 148):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

C

(Human IL-21)

CAGGGCC AGGACAGGC ACATGATCCGGATGAGGC AGCTCATCGAC ATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCC AGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGA GCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAG GAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAAC GTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The second chimeric polypeptide of IL-2l/IL-l5Ra sushi domain (including leader sequence) is as follows (SEQ ID NO: 147):

(Signal Sequence)

MKWVTFISLLFLF S SAYS

(Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKS ANTGNNERIINV SIKKLKRKPP STNAGRRQKHRLTCP SCD S YEKKPPKEFLER FK SLLQKMIHQHL S SRTHGSED S (Human IL-15Ra sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRKAGTS SLTECVLNKATNVAH WTTPSLKCIR

Example 70: Creation of an IL-7/TF/IL-15 DNA construct

In a non-limiting example, an exemplary first chimeric polypeptide of IL-

7/TF/IL-15 was made by linking the IL-7 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-7/TF construct with the N-terminus coding region of IL-15. The nucleic acid sequence encoding the first chimeric polypeptide of IL-7/TF/IL-15 (including leader sequence), synthesized by Genewiz, is as follows (SEQ ID NO: 144):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC

C

(Human IL-7 fragment)

GATTGCGACATCGAGGGC AAGGACGGC AAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GC AGAAGAAGCTGAACGACCTGT GCTTCCTGAAGAGGCTGCTGC AGGAGAT C AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT

(Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACC ACCGAC ACCGAGT GCGATCTC ACCGAT GAGATCGT GAAAGAT GT GAAAC AGA CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACAC CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT GC AT GGGC C A AGA A A AGGGC GAGTTCC GGGAG

(Human IL-15)

AACTGGGT GAACGT CAT C AGCGATTT AAAGAAGATCGA AGATTT A ATT C AGT

CCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGT

AAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGA

GAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCC

AATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGT

GCGA AGAGCTGGAGGAGAAGAAC ATC AAGGAGTTTCTGC AATCCTTT GT GCA

CATTGTCCAGATGTTCATCAATACCTCC

The first chimeric polypeptide of IL-7/TF/IL-15 (including leader sequence), is as follows (SEQ ID NO: 143):

(Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A ALGE AQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKCF YTTD TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQ SFEQ V GTK VN VT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYWK S S S SG KKT AKTNTNEFLID VDKGEN Y CF S VQ A VIP SRT VNRK S TD SP VECMGQEKGEFR

E

(Human IL-15) NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI SLE SG

DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN

TS

Example 71: Secretion of IL-21/IL-15RaSu and IL-7/TF/IL-15 fusion proteins

The IL-2l/IL-l5RaSu and IL-7/TF/IL-15 DNA constructs were cloned into a pMSGV-l modified retrovirus expression vector (as described by Hughes, Hum Gene Ther 16:457-72, 2005, hereby incorporated by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of a soluble IL-7/TF/IL-l5:IL-2l/IL-l5RaSu protein complex (referred to as 7tl5-2ls). The 7tl 5-21 s protein was purified from CHO-K1 cell culture supernatant using anti-TF antibody (IgGl) affinity chromatography and size exclusion chromatography resulting in soluble (non-aggregated) protein complexes consisting of IL-2l/IL-l5RaSu and IL-7/TF/IL-15 fusion proteins. See, Figures 82 and 83. Example 72: Analytical size exclusion chromatography (SEC) analysis of IL-21/IL- 15RaSu and IL-7/TF/IL-15 fusion proteins

To determine if anti-tissue factor monoclonal antibody and 7tl 5-21 s can form an antibody-fusion-molecule complex, analytical size exclusion chromatography (SEC) was performed. A Superdex 200 Increase 10/300 GL gel filtration column (from GE

Healthcare) was connected to an AKTA Avant system (from GE Healthcare). The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min.

Samples of the anti-TF mAh (1 mg/mL), 7tl5-2ls (lmg/mL), and a mixture of combined at a 1 : 1 ratio, so the final concentration of each protein is 0.5mg/mL) were in PBS. Each sample was injected into the Superdex 200 column using a capillary loop, and analyzed by SEC. The SEC chromatograph of each sample was shown in Figure 84. The SEC results indicated that there are two protein peaks for 7tl 5-21 s, likely representing a dimer

(with an apparent molecular weight of l99.2kDa) and a higher oligomer of 7tl 5-21 s, and there is one peak (with an apparent molecular weight of 206.8kDa) for the anti-TF mAh.

However, as expected, a new protein peak with a higher molecular weight (with an apparent molecular weight of 576.9 kDa) was formed in the mixture sample containing the anti-TF mAb and 7tl 5-21 s, indicating that the anti-TF mAh and 7tl 5-21 s form an antibody-antigen complex through the binding of anti-TF mAb to TF in the fusion protein complex.

Example 73: Expansion capacity of primary natural killer (NK) cells by 7tl5-21s complex + anti-TF IgGl antibody

To assess the 7tl 5-21 s complex’s ability to expand primary natural killer (NK) cells, 7tl5-2ls complex and 7tl5-2ls complex + anti-TF IgGl antibody are added to K cells obtained from samples of fresh human leukocytes. Cells are stimulated with 50nM of 7tl 5-21 s complex with or without 25 nM of anti-TF IgGl or anti-TF IgG4 antibody at 37°C and 5% CO2. Cells are maintained at concentration at 0.5 x l0 ⁶ /mL not exceeding

2.0 x l0 ⁶ /mL by counting every 48-72 hours and media is replenished with fresh stimulator. Cells stimulated with 7tl5-2ls complex or anti-TF IgGl antibody or anti-TF IgG4 antibody or anti-TF IgG4 + 7tl 5-21 s complex are maintained up to day 5.

Expansion of primary NK cells upon incubation with 2ltl5-7s complex + anti-TF IgGl antibody is observed.

Example 74: Activation of expanded NK cells by the 7tl5-21s complex + anti-TF IgGl antibody

Primary NK cells are induced ex vivo following overnight stimulation of purified NK cells with 7tl5-2ls complex + anti-TF IgGl antibody. Fresh human leukocytes are obtained from a blood bank and CD56+ NK cells are isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells is >80% and is confirmed by staining with CD56-BV421 and CD16-BV510 specific antibodies

(BioLegend). Cells are counted and resuspended in 1 x l0 ⁶ /mL in a 24 well flat bottom plate in 1 mL of complete media (RPMI 1640 (Gibco), supplemented with 4 mM L- glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), non-essential amino acid (Thermo Life Technologies), sodium pyruvate (Thermo Life Technologies), and 10% FBS (Hyclone)). Cells are stimulated with 50 nM of 7tl 5-21 s with or without 25 nM of anti-TF IgGl antibody at 37°C and 5% CO2. Cells are counted every 48-72 hours and maintained at a concentration of 0.5 x l0 ⁶ /mL to 2.0 x l0 ⁶ /mL until day 14. Media is periodically replenished with fresh stimulator. Cells are harvested and surface stained at day 3 with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies

(Biolegend and analyzed by Flow Cytometry-Celeste-BD Bioscience). The activation marker CD25 MFI are observed to increase with 7tl 5-21 s complex + anti-TF IgGl antibody stimulation, but not 7tl5-2ls complex stimulation. The activation marker CD69 MFI is observed to increase with both 7tl 5-21 s complex + anti-TF IgGl antibody and with 7tl 5-21 s complex, alone.

Example 75: 7tl5-16s21 fusion protein generation and characterization

A fusion protein complex was generated comprising of anti-CD l6scFv/IL- l5RaSu/IL-2l and IL-7/TF/IL-15 fusion proteins. The human IL-7 and IL-21 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking the IL-7 sequence to the N- terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15. See, Figures 85 and 86.

The nucleic acid and protein sequences of a construct comprising IL-7 linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the IL-7/TF/IL-15 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human IL-7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT

GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AG AT C AAGACCTGCTGGAAC AAGATCCTGATGGGC ACC AAGGAGC AT

(Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of IL-7/TF/IL-15 fusion protein (including the leader sequence) is as follows:

( Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-7) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A AL GE AQPTK SLEENK SLKEQKKLNDLCFLKRLLQEIKT C WNKILMGTKEH

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC

FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS Constmcts were also made by linking the anti-CD l6scFv sequence to the N- terminus coding region of IL-l5RaSu chain followed by the N-terminus coding region of IL-21 which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the anti-CDl6scFv linked to the N-terminus of IL-l5RaSu chain followed by the N-terminus coding region of IL-21 are shown below.

The nucleic acid sequence of the anti-CD l6SscFv/IL- 15 RaSu/IL-2l construct

(including signal peptide sequence) is as follows:

( Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

((Anti-human CD scFv)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACC GTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGT ACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAA CAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCG CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTG CAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAG CTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGA GG AGG AT C C G AGGT GC AGC T GGT GG AGT C C GG AGG AGG AGT GGT G AGGC C T GGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTA CGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCC GGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCA GGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAA CTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCC CTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

(Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCC A AGGAGTTCCTGGAGAGGTTC AAGTCCCTGCTGC AGAAGAT GAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence of the anti-CD l6scFv/IL-l 5RaSu/IL-2l construct (including signal peptide sequence) is as follows:

( Signal peptide)

MKWVTFISLLFLF S SAYS

(Anti-human CD scFv)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRP S GIPDRF S GS S S GNT ASLTIT GAQ AEDE AD Y Y CN SRD S S GNH V VF GGGTKL TV GHGGGGS GGGGS GGGGSE V QL VES GGGVVRPGGSLRL S C A ASGF TFDD Y GM SWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLR AEDTAVYY C ARGRSLLFDYW GQGTLVT V SR

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNK AT NVAHWTTPSLKCIR

(Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEW S AF SCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHL S SRTHGSED S

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The anti-CD l6scFv/IL-l5RaSu/IL-2l and IL-7/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble IL-7/TF/IL-l5:anti-CDl6scFv/IL-l5RaSu/IL-2l protein complex (referred to as 7tl5-l6s2l), which can be purified by anti-TF antibody- based affinity and other chromatography methods.

Binding of 7tl5-16s21 to CHO cells expressing human CD 16b

CHO cells were transfected with human CDl6b in a pMC plasmid and selected with 10 pg/mL of blasticidin for 10 days. The CHO cells stably expressing CDl6b were stained with 1.2 pg/mL of 7tl5-l6s2l (containing anti-human CD16 scFv) or 18tl 5-l2s (does not contain anti-human CD 16 scFv) as a negative control, and then stained with biotinylated anti-human tissue factor and PE conjugated streptavidin. Only anti-human CDl6scFv containing 7tl5-l6s2l stained the cells as shown in Figure 87A. l8tl5-l2s did not stain the CHO cells expressing human CDl6b as showed in Figure 87B. Detection ofIL-15, II -21, andIL-7 in 7tl5-16s21 using ELISA

A 96-well plate was coated with 100 pL (8 pg/mL) of anti-TF antibody in R5 (coating buffer) and incubated at room temperature (RT) for 2 hrs. The plates were washed 3 times and blocked with 100 pL of 1% BSA in PBS. Serial dilution of 7tl 5- l6s2l (at a 1 :3 ratio) were added to the wells, and incubated at RT for 60 min. Following 3 washes, 50 ng/mL of biotinylated-anti-ILl5 antibody (BAM247, R&D Systems), 500 ng/mL of biotinylated-anti-IL-2l antibody (13-7218-81, R&D Systems), or 500 ng/mL of biotinylated-anti-IL-7 antibody (506602, R&D Systems) was added to the wells and incubated at RT for 60 min. The plate was washed 3 times, and incubated with 0.25 pg/mL of HRP-SA (Jackson ImmunoResearch) at 100 pL per well for 30 min at RT, followed by 4 washes and incubation with 100 pl of ABTS for 2 mins at RT.

Absorbance was read at 405 nm. As shown in Figures 88A-88C, the IL-15, IL-21, and IL-7 domains in 7tl5-l6s2l were detected by the individual antibodies. The IL-15 in 7tl5-16s21 promotes II-2R[ _> and common g chain containing 32Όb cell proliferation

To analyze the activity of IL-15 in 7tl 5- 16s21 , the IL-15 activity of 7tl5-l6s2l was compared to recombinant IL-15 using 32ϋb cells that express IL2RP and common g chain, and evaluating their effects on promoting cell proliferation. IL-15 dependent 32ϋb cells were washed 5 times with IMDM-l0% FBS and seeded in the wells at 2 x 10 ⁴ cells/well. Serially-diluted 7tl5-l6s2l or IL-15 were added to the cells (Figure 89).

Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by adding 10 pl of WST1 to each well on day 3 and incubating for an additional 3 hours in a CO2 incubator at 37°C. The absorbance at 450 nm was measured by analyzing the amount of formazan dye produced. As shown in Figure 5, 7tl5-l6s2l and IL-15 promoted 32ϋb cell proliferation, with the EC50 of 7tl5-l6s2l and IL-15 being 172.2 pM and 16.63 pM, respectively.

Purification elution chromatograph of 7tl5-16s21 from anti-TF affinity column 7tl5-l6s2l harvested from cell culture was loaded onto the anti-TF affinity column equilibrated with 5 column volumes of PBS. The column was then washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elution peak was collected and neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. Figure 90 is a line graph showing the chromatographic profile of 7tl5-l6s2l protein containing cell culture supernatant following binding and elution on anti-TF antibody resin. As shown in Figure 90, the anti- TF antibody affinity column bound 7tl5-l6s2l which contains TF. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min. Analytical size exclusion chromatography (SEC) analysis of 7tl5-16s21

To perform size exclusion chromatography (SEC) analysis for 7tl5-l6s2l, a Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare) connected to an AKTA Avant system (GE Healthcare) was used. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. A sample containing 7tl5-l6s2l in PBS was injected into the Superdex 200 column using a capillary loop, and analyzed by SEC. As shown in Figure 91, the SEC results showed two protein peaks for 7tl 5- 16s21.

Example 76: TGFRtl5-16s21 fusion protein generation and characterization

A fusion protein complex was generated comprising anti -human CDl6scFv/IL- l5RaSu/IL2l and TGFP Receptor IETF/IL-15 fusion proteins (Figure 92 and 93). The human TGFp Receptor II (Ile24-Aspl59), tissue factor 219, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking two TGFp Receptor II sequences with a G4S(3) linker to generate a single chain version of TGFp Receptor II and then directly linking to the N-terminus coding region of tissue factor 219 followed by the N- terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising two TGFp Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the two TGFp Receptor II/TF/IL-15 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Two Human ΊCiIb Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGT GC AGA AGAGCGT GAAT AATGAC AT GATCGTGACCGAT AAC AATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC

(Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG

CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT

ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA

ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC

ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA

CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC

ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC

AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT

GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA

AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC

AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT

T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFP Receptor II/TF/IL-15 fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human KIIb Receptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLET V CHDPKLP YHDFILED A A SPKCIMKEKKKPGETFFMC S C S SDE CNDNIIF SEEYNT SNPD (Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS

Constmcts were also made by attaching anti-human CDl6scFv directly linking to the N-terminus coding region of IL-l5RaSu chain followed by the N-terminus coding region of IL-21 which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the anti-human CDl6scFv linked to the N-terminus of IL-l5RaSu followed by the N-terminus coding region of IL-21 are shown below.

The nucleic acid sequence of the anti-CD l6scFv/IL- 15 RaSu/IL-2l construct (including signal peptide sequence) is as follows:

( Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Anti-human CD scFv)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACC GTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGT ACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAA CAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCG CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTG CAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAG CTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGA GG AGG AT C C G AGGT GC AGC T GGT GG AGT C C GG AGG AGG AGT GGT G AGGC C T GGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTA CGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCC GGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCA GGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAA CTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCC CTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

(Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCC AAGGAGTTCCTGGAGAGGTTC AAGTCCCTGCTGC AGAAGAT GAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence of the anti-CD l6scFv/IL-l 5RaSu/IL-2l construct (including signal peptide sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Anti-human CD scFv)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRP S GIPDRF S GS S S GNT ASLTIT GAQ AEDE AD Y Y CN SRD S S GNH V VF GGGTKL TV GHGGGGS GGGGS GGGGSE V QL VES GGGVVRPGGSLRL S C A ASGF TFDD Y GM SWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLR AEDTAVYY C ARGRSLLFDYW GQGTLVT V SR

(Human IL-15R a sushi domain) ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNK AT NVAHWTTPSLKCIR

(Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEW S AF SCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHL S SRTHGSED S

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The anti-CD 16scFv/IL- 15RaSu/IL-21 and TGFR/TF/IL- 15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble TGFR/TF/IL-l5:CDl6scFv/IL- l5RaSu/IL-2l protein complex (referred to as TGFRtl5-l6s2l), which can be purified by anti-TF antibody -based affinity and other chromatography methods. Interaction between TGFRtl5-16s21 and CHO cells expressing human CD 16b

CHO cells were transfected with human CDl6b in a pMC plasmid and selected with 10 pg/mL of blasticidin for 10 days. Cells stably expressing CDl6b were stained with 1.2 pg/mL of TGFRtl5-l6s2l (containing anti -human CD 16 scFv) or 7tl 5-21 s (no anti-human CD 16 scFv) as a negative control, and with biotinylated anti -human tissue factor antibody and PE conjugated streptavidin. As shown in Figures 94A and 94B,

TGFRtl5-l6s2l, which contains anti-human CDl6scFv, showed positive binding, while 7tl 5-21 s did not show binding.

Effect of TGFRtl5-16s21 on TGFfl activity in HEK-Blue TGFf cells

To evaluate the activity of TGFpRII in TGFRtl5-l6s2l, the effect of TGFRtl5-

!6s2l on the activity of TGFpl in HEK-Blue TGFP cells was analyzed. HEK-Blue TGFP cells (Invivogen) were washed twice with pre-warmed PBS and resuspended in the testing medium (DMEM, 10% heat-inactivated FCS, lx glutamine, lx anti-anti, and 2x glutamine) at 5 x 10 ⁵ cells/mL. In a flat-bottom 96-well plate, 50 mΐ cells were added to each well (2.5 x 10 ⁴ cells/well) and followed with 50 pL O.lnM TGFp i (R&D systems). TGFRtl5-l6s2l or TGFR-Fc (R&D Systems) prepared at a 1 :3 serial dilution was then added to the plate to reach a total volume of 200 pL. After 24 hrs of incubation at 37°C, 40 pL of induced HEK-Blue TGFp cell supernatant was added to 160 pL pre-warmed QUANTI-Blue (Invivogen) in a flat-bottom 96-well plate, and incubated at 37°C for 1-3 hrs. The OD values were then determined using a plate reader (Multiscan Sky) at 620-655 nM. The ICso of each protein sample was calculated with GraphPad Prism 7.04. The IC5 ₀ of TGFRtl5-l6s2l and TGFR-Fc were 9127 pM and 460.6 pM respectively. These results showed that the TGFpRII domain in TGFRtl5-l6s2l was able to block the activity of TGFP-l in HEK-Blue TGFP cells. See, Figure 95. The IL-15 in TGFRtl5-16s21 promotes II -2R[ _> and common g chain containing

32Όb cell proliferation

To analyze the activity of IL-15 in TGFRtl5-l6s2l, the IL-15 activity of

TGFRtl5-l6s2l was compared to recombinant IL-15 using 32ϋb cells that express IL2RP and common g chain, and evaluating their effects on promoting cell proliferation. IL-15 dependent 32ϋb cells were washed 5 times with IMDM-l0% FBS and seeded in the wells at 2 x 10 ⁴ cells/well. Serially-diluted TGFRtl5-l6s2l or IL-15 were added to the cells (Figure 96). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by adding 10 pL of WST1 to each well on day 3 and incubating for an additional 3 hours in a CO2 incubator at 37°C. The absorbance at 450 nm was measured by analyzing the amount of formazan dye produced. As shown in

Figure 96, TGFRtl5-l6s2l and IL-15 promoted 32ϋb cell proliferation, with the EC50 of TGFRtl5-l6s2l and IL-15 being 51298 pM and 10.63 pM, respectively.

Detection of IL-15, IL-21, and TGI lRII in TGFRtl5-16s21 using ELISA

A 96-well plate was coated with 100 pL (8 pg/mL) of anti-TF antibody in R5 (coating buffer) and incubated at room temperature (RT) for 2 hrs. The plates were washed 3 times and blocked with 100 pL of 1% BSA in PBS. TGFRtl5-l6s2l serially diluted at a 1 :3 ratio was added and incubated at RT for 60 min. Following three washes, 50 ng/mL of biotinylated-anti-IL-l5 antibody (BAM247, R&D Systems), 500 ng/mL of biotinylated-anti-IL-2l antibody (13-7218-81, R&D Systems), or 200 ng/mL of biotinylated-anti-TGFpRII antibody (BAF241, R&D Systems) was applied per well, and incubated at RT for 60 min. Following three washes, incubation with 0.25 pg/mL of HRP-SA (Jackson ImmunoResearch at 100 pL per well for 30 min at RT was carried out, followed by 4 washes and incubation with 100 pL of ABTS for 2 mins at RT.

Absorbance was read at 405 nm. As shown in Figures 97A-97C, the IL-15, IL-21, and TGFpRII domains in TGFRtl5-l6s2l were detected by the respective antibodies.

Purification elution chromatograph ofTGFRtl5-16s21 using anti-TF antibody affinity column

TGFRtl5-l6s2l harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in Figure 14, the anti-TF antibody affinity column bound to TGFRtl5- l6s2l which contains tissue factor as a fusion partner. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Reduced SDS-PAGE o/TGFRtl5-16s21

To determine the purity and molecular weight of the TGFRtl5-l6s2l protein, protein sample purified with anti-TF antibody affinity column (Figure 98) was analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel)

electrophoresis (SDS-PAGE) under reduced condition. After electrophoresis, the gel was stained with InstantBlue for about 30 min, followed by destaining overnight in purified water.

To verify that the TGFRtl5-l6s2l protein undergoes glycosylation after translation in CHO cells, a deglycosylation experiment was conducted using the Protein Deglycosylation Mix II kit from New England Biolabs according to the manufacturer’s instructions. Figure 99 shows results from the reduced SDS-PAGE analysis of the sample in non-deglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state. The results showed that the TGFRtl5-l6s2l protein is glycosylated when expressed in CHO cells. After deglycosylation, the purified sample showed expected molecular weights (69 kDa and 48 kDa) in the reduced SDS gel. Lane M was loaded with lOpL of SeeBlue Plus2 Prestained Standard.

Example 77: 7tl5-7s fusion protein generation and characterization

A fusion protein complex was generated comprising IL-7/TF/IL-15 and IL-7/IL- l5RaSu fusion proteins. The human IL-7, tissue factor 219, and IL-15 sequences were obtained from the ETniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking the IL-7 sequence to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising IL-7 linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of 7tl5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA

AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA

GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC

ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG

CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA

AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG

AG AT C AAGACCTGCTGGAAC AAGATCCTGATGGGC ACC AAGGAGC AT

(Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC The amino acid sequence of 7tl 5 fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A AL GE AQPTK SLEENK SLKEQKKLNDLCFLKRLLQEIKT C WNKILMGTKEH

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS

Constructs were also made by linking the IL-7 sequence to the N-terminus coding region of IL-l 5RaSu chain which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the IL-7 linked to the N-terminus of IL- l 5RaSu chain are shown below.

The nucleic acid sequence of 7s construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT

GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AG AT C AAGACCTGCTGGAAC AAGATCCTGATGGGC ACC AAGGAGC AT

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTAC AGCCGGGAGAGGTATATCTGTAACAGCGGCTTC A AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence of 7s fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A AL GE AQPTK SLEENK SLKEQKKLNDLCFLKRLLQEIKT C WNKILMGTKEH

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNKAT NVAHWTTPSLKCIR

The IL-7/TF/IL-15 and IL-7/IL-l5RaSu constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Hier 2005; 16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble IL-7/TF/IL-l5:IL-7/IL-l5RaSu protein complex (referred to as 7tl5-7s), which can be purified by anti-TF antibody affinity and other chromatography methods. See, Figures 100 and 101.

Purification elution chromatograph of 7tl5-7s using anti-TF antibody affinity column

7tl5-7s harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in Figure 102, the anti-TF antibody affinity column bound to 7tl5-7s which contains tissue factor (TF) as a fusion partner. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti- TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except the elution step, which was 2 mL/min.

Immunostimulation of 7tl5-7s in C57BL/6 mice

7tl5-7s is a multi-chain polypeptide (a type A multi-chain polypeptide described herein) that includes the first polypeptide that is a soluble fusion of human IL-7, human tissue factor 219 fragment and human IL-15 (7tl 5), and the second polypeptide that is a soluble fusion of human IL-7 and sushi domain of human IL-15 receptor alpha chain (7s).

CHO cells were co-transfected with the IL7-TF-IL15 (7tl 5) and åL7-ILl5Ra sushi domain (7s) vectors. The 7tl5-7s complex was purified from the transfected CHO cell culture supernatant. The IL-7, IL-15 and tissue factor (TF) components were demonstrated in the complex by ELISA as shown in Figure 103. A humanized anti-TF monoclonal antibody was used as the capture antibody to determine TF in 7tl5-7s, and biotinylated anti-human IL-15 antibody (R&D systems) and biotinylated anti-human IL-7 antibody (R&D Systems) were used as the detection antibodies to respectively detect IL- 15 and IL-7 in 7tl5-7s, followed by peroxidase conjugated streptavidin (Jackson

ImmunoResearch Lab) and ABTS substrate (Surmodics IVD, Inc.). 7tl5-7s was subcutaneously injected into C57BL/6 mice at 10 mg/kg to determine the

immunostimulatory activity of 7tl5-7s in vivo. C57BL/6 mice subcutaneously treated with PBS were used as control. The mouse spleens were collected and weighed day 4 post treatment. Single splenocytes suspensions were prepared, and with fluorochrome- conjugated anti-CD4, anti-CD8, and anti-NKl.l antibodies and the percentage of CD4 ⁺

T cells, CD8 ⁺ T cells, and NK cells was analyzed by flow cytometry. The results showed that 7tl5-7s was effective at expanding splenocytes based on spleen weight (Figure 104A) and specifically, the percentages of CD8 ⁺ T cells and NK cells were higher compared to control -treated mice (Figure 104B).

Example 78: TGFRtl5-TGFRs fusion protein generation and characterization

A fusion protein complex was generated comprising of TGFP Receptor II/IL- l5RaSu and TGFp Receptor II/TF/IL- l 5 fusion proteins. The human TGFp Receptor II (Ile24-Aspl59), tissue factor 219, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking two TGFP Receptor II sequences with a G4S(3) linker to generate a single chain version of TGFP Receptor II and then directly linking to the N- terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising two TGFP Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the two TGFp Receptor II/TF/IL-l 5 construct

(including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Two Human ΊCiIb Receptor II fragments) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGT GC AGA AGAGCGT GAAT AATGAC AT GATCGTGACCGAT AAC AATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC

(Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG (Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFP Receptor II/TF/IL-15 fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human KIIb Receptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLET V CHDPKLP YHDFILED A A SPKCIMKEKKKPGETFFMC S C S SDE CNDNIIF SEEYNT SNPD

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS Constructs were also made by attaching two TGFp Receptor II directly to the IL- l5RaSu chain which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the TGFP Receptor II linked to the N-terminus of IL-l5RaSu are shown below.

The nucleic acid sequence of the TGFp Receptor II/IL-15 RaSu construct (including signal peptide sequence) is as follows:

{Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Two human ΊCiIb Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGT GC AGA AGAGCGT GAAT AATGAC AT GATCGTGACCGAT AAC AATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG

AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA

CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence of the two TGFp Receptor II/IL-l5RaSu construct (including signal peptide sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Two human KIIb Receptor II extra-cellular domains)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVC VAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK

PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLET V CHDPKLP YHDFILED A A SPKCIMKEKKKPGETFFMC S C S SDE CNDNIIF SEEYNT SNPD

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNK AT NVAHWTTPSLKCIR

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The TGFpR/IL- l 5RaSu and TGFpR/TF/IL- 15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO- Kl cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble TGFpR/TF/IL- 15:TGFpR/IL- 15RaSu protein complex (referred to as TGFRtl5-TGFRs), which can be purified by anti-TF antibody affinity and other chromatography methods. See, Figures 105 and 106. Effect of TGFRtl 5-TGFRs on TGFfl activity in HEK-Blue TGFf cells

To evaluate the activity of TGFpRII in TGFRtl 5-TGFRs, the effect of TGFRtl 5- l6s2l on the activity of TGFpl in HEK-Blue TGFP cells was analyzed. HEK-Blue TGFP cells (Invivogen) were washed twice with pre-warmed PBS and resuspended in the testing medium (DMEM, 10% heat-inactivated FCS, lx glutamine, lx anti-anti, and 2x glutamine) at 5 x 10 ⁵ cells/mL. In a flat-bottom 96-well plate, 50 pL cells were added to each well (2.5 x 10 ⁴ cells/well) and followed with 50 pL O.lnM TGFp i (R&D systems). TGFRtl5-l6s2l or TGFR-Fc (R&D Systems) prepared at a 1 :3 serial dilution was then added to the plate to reach a total volume of 200 pL. After 24hrs of incubation at 37°C, 40 pL of induced HEK-Blue TGFp cell supernatant was added to 160 pL pre-warmed

QUANTI-Blue (Invivogen) in a flat-bottom 96-well plate, and incubated at 37°C for 1-3 hrs. The OD values were then determined using a plate reader (Multiscan Sky) at 620-655 nM. See, Figure 107. The ICso of each protein sample was calculated with GraphPad Prism 7.04. The ICso of TGFRtl 5-TGFRs and TGFR-Fc were 216.9 pM and 460.6 pM respectively. These results showed that the TGFpRII domain in TGFRtl 5-TGFRs was able to block the activity of TGFpl in HEK-Blue TGFp cells.

The IL-15 in TGFRtl 5-TGFRs promotes II -2R[ _> and common g chain containing 32 Iff cell proliferation

To evaluate the activity of IL-15 in TGFRtl 5-TGFRs, the IL-15 activity of

TGFRtl 5-TGFRs was compared to recombinant IL-15 using 32ϋb cells that express IL2RP and common g chain, and evaluating their effects on promoting cell proliferation. IL-15 dependent 32ϋb cells were washed 5 times with IMDM-l0% FBS and seeded in the wells at 2 x 10 ⁴ cells/well. Serially-diluted TGFRtl5-TGFRs or IL-15 were added to the cells (Figure 108). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by adding 10 pL of WST1 to each well on day 3 and incubating for an additional 3 hours in a CO2 incubator at 37°C. The absorbance at 450 nm was measured by analyzing the amount of formazan dye produced. As shown in Figure 108, TGFRtl 5-TGFRs and IL-15 promoted 32ϋb cell proliferation, with the EC50 of TGFRtl5-l6s2l and IL-15 being 1901 pM and 10.63 pM, respectively. Detection ofIL-15 and I ^' GIfiRII domains in TGFRtl 5-TGFRs with corresponding antibodies using ELISA

A 96-well plate was coated with 100 pL (8 pg/mL) of anti-TF antibody in R5 (coating buffer) and incubated at room temperature (RT) for 2 hrs. The plates were washed 3 times and blocked with 100 pL of 1% BSA in PBS. TGFRtl5-TGFRs was added at a 1 :3 serial dilution, and incubated at RT for 60 min. After 3 washes, 50 ng/mL of biotinylated-anti-IL-l5 antibody (BAM247, R&D Systems), or 200 ng/mL of biotinylated-anti-TGFpRII antibody (BAF241, R&D Systems) was added to the wells and incubated at RT for 60 min. Next the plates were washed 3 times, and 0.25 pg/mL of HRP-SA (Jackson ImmunoRe search) at 100 pL per well was added and incubated for 30 min at RT, followed by 4 washes and incubation with 100 pL of ABTS for 2 mins at RT. Absorbance at 405 nm was read. As shown in Figure 109A and 109B, the IL-15 and TGFpRII domains in TGFRtl 5-TGFRs were detected by the individual antibodies. Purification elution chromatograph of TGFRtl 5-TGFRs from anti-TF antibody affinity column

TGFRtl 5-TGFRs harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in Figure 110, the anti-TF antibody affinity column bound to TGFRtl 5-TGFRs which contains TF as a fusion partner. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min. Analytical size exclusion chromatography (SEC) analysis of TGFRtl 5-TGFRs A Superdex 200 Increase 10/300 GL gel filtration column (from GE Healthcare) was connected to an AKTA Avant system (from GE Healthcare). The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. A sample containing TGFRtl5-TGFRs in PBS was injected into the Superdex 200 column using a capillary loop, and analyzed by SEC. The SEC chromatograph of the sample is shown in Figure 111. The SEC results showed four protein peaks for TGFRtl 5-TGFRs

Reduced SDS-PAGE analysis of TGFRtl 5-TGFRs

To determine the purity and molecular weight of the TGFRtl 5-TGFRs protein, protein sample purified with anti-TF antibody affinity column was analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS- PAGE) method under reduced condition. After electrophoresis, the gel was stained with InstantBlue for about 30 min, followed by destaining overnight in purified water.

To verify that the TGFRtl 5-TGFRs protein undergoes glycosylation after translation in CHO cells, a deglycosylation experiment was conducted using the Protein Deglycosylation Mix II kit from New England Biolabs and the manufacturer’s instructions. Figure 112 shows the reduced SDS-PAGE analysis of the sample in non- deglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state. The results showed that the TGFRtl 5-TGFRs protein is glycosylated when expressed in CHO cells. After deglycosylation, the purified sample showed expected molecular weights (69 kDa and 39 kDa) in the reduced SDS gel. Lane M was loaded with 10 ul of SeeBlue Plus2 Prestained Standard. Immunostimulatory activity of TGFRtl 5-TGFRs in C57BL/6 mice

TGFRtl 5-TGFRs is a multi-chain polypeptide (a type A multi-chain polypeptide described herein) that includes a first polypeptide that is a soluble fusion of two TGFpRII domains, human tissue factor 219 fragment and human IL-15, and the second polypeptide that is a soluble fusion of two TGFpRII domains and sushi domain of human IL-15 receptor alpha chain. Wild type C57BL/6 mice were treated subcutaneously with either control solution or with TGFRtl5-TGFRs at a dosage of 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10 mg/kg. Four days after treatment, spleen weight and the percentages of various immune cell types present in the spleen were evaluated. As shown in Figure 113 A, the spleen weight in mice treated with TGFRtl 5-TGFRs increased with increasing dosage of TGFRtl 5-

TGFRs. Moreover, the spleen weight in mice treated with 1 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRtl 5-TGFRs were higher as compared to mice treated with the control solution, respectively. In addition, the percentages of CD4 ⁺ T cells, CD8 ⁺ T cells, NK cells, and CDl9 ⁺ B cells present in the spleen of control -treated and TGFRtl 5-TGFRs - treated mice were evaluated. As shown in Figure 113B, in the spleens of mice treated with TGFRtl 5-TGFRs, the percentages of CD8 ⁺ T cells and NK cells both increased with increasing dosage of TGFRtl 5-TGFRs. Specifically, the percentages of CD8 ⁺ T cells were higher in mice treated with 0.3 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRtl 5-TGFRs compared to control-treated mice, and the percentages of NK cells were higher in mice treated with 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRtl 5-TGFRs compared to control -treated mice. These results demonstrate that TGFRtl 5-TGFRs is able to stimulate immune cells in the spleen, in particular CD8 ⁺ T cells and NK cells.

The pharmacokinetics of TGFRtl 5-TGFRs molecules were evaluated in wild type C57BL/6 mice. The mice were treated subcutaneously with TGFRtl 5-TGFRs at a dosage of 3 mg/kg. The mouse blood was drained from tail vein at various time points and the serum was prepared. The TGFRtl 5-TGFRs concentrations in mouse serum was determined with ELISA (capture: anti-human tissue factor antibody; detection:

biotinylated anti-human TGFp receptor antibody and followed by peroxidase conjugated streptavidin and ABTS substrate). The results showed that the half-life of TGFRtl 5- TGFRs was 12.66 hours in C57BL/6 mice.

The mouse splenocytes were prepared in order to evaluate the immunostimulatory activity of TGFRtl 5-TGFRs over time in mice. As shown in Figure 114A, the spleen weight in mice treated with TGFRtl 5-TGFRs increased 48 hours posttreatment and continued to increase over time. In addition, the percentages of CD4 ⁺ T cells, CD8 ⁺ T cells, NK cells, and CDl9 ⁺ B cells present in the spleen of control -treated and TGFRtl 5- TGFRs-treated mice were evaluated. As shown in Figure 114B, in the spleens of mice treated with TGFRtl5-TGFRs, the percentages of CD8 ⁺ T cells and NK cells both increased at 48 hours after treatment and were higher and higher overtime after the single dose treatment. These results further demonstrate that TGFRtl5-TGFRs is able to stimulate immune cells in the spleen, in particular CD8 ⁺ T cells and NK cells.

Furthermore, the dynamic proliferation of immune cells based on Ki67 expression of splenocytes and cytotoxicity potential based on granzyme B expression were evaluated in splenocytes isolated from mice following a single dose (3 mg/kg) of TGFRtl5-TGFRs. As shown in Figure 115A and 115B, in the spleens of mice treated with TGFRtl5- TGFRs, the expression of Ki67 and granzyme B by NK cells increased at 24 hours after treatment and its expression of CD8 ⁺ T cells and NK cells both increased at 48 hours and later time points after the single dose treatment. These results demonstrate that

TGFRtl5-TGFRs not only increases the numbers of CD8 ⁺ T cells and NK cells but also enhance the cytotoxicity of these cells. The single dose treatment of TGFRtl5-TGFRs led CD8 ⁺ T cells and NK cells to proliferate for at least 4 days.

The cytotoxicity of the splenocytes from TGFRtl5-TGFRs -treated mice against tumor cells was also evaluated. Mouse Moloney leukemia cells (Yac-l) were labeled with CellTrace Violet and were used as tumor target cells. Splenocytes were prepared from TGFRtl5-TGFRs (3 mg/kg)-treated mouse spleens at various time points post treatment and were used as effector cells. The target cells were mixed with effector cells at an E:T ratio = 10: 1 and incubated at 37°C for 20 hours. Target cell viability was assessed by analysis of propidium iodide positive, violet-labeled Yac-l cells using flow cytometry. Percentage of Yac-l tumor inhibition was calculated using the formula, (1- [viable Yac-l cell number in experimental sample]/[viable Yac-l cell number in the sample without splenocytes]) x 100. As shown in Figure 116, splenocytes from

TGFRtl5-TGFRs -treated mice had stronger cytotoxicity against Yac-l cells than the control mouse splenocytes.

Tumor size analysis in response to chemotherapy and/or TGFRtl 5-TGFRs Pancreatic cancer cells (SW1990, ATCC® CRL-2172) were subcutaneously (s.c.) injected into C57BL/6 scid mice (The Jackson Laboratory, 001913, 2xl0 ⁶ cells/mouse, in lOOpL HBSS) to establish the pancreatic cancer mouse model. Two weeks after tumor cell injection, chemotherapy was initiated in these mice intraperitoneally with a combination of Abraxane (Celgene, 68817-134, 5 mg/kg, i.p.) and Gemcitabine (Sigma Aldrich, G6423, 40 mg/kg, i.p.), followed by immunotherapy with TGFRtl5-TGFRs (3 mg/kg, s.c.) in 2 days. The procedure above was considered one treatment cycle and was repeated for another 3 cycles (1 cycle/week). Control groups were set up as the SW1990- injected mice that received PBS, chemotherapy (Gemcitabine and Abraxane), or

TGFRtl5-TGFRs alone. Along with the treatment cycles, tumor size of each animal was measured and recorded every other day, until the termination of the experiment 2 months after the SW1990 cells were injected. Measurement of the tumor volumes were analyzed by group and the results indicated that the animals receiving a combination of chemotherapy and TGFRtl5-TGFRs had significantly smaller tumors comparing to the PBS group, whereas neither chemotherapy nor TGFRtl5-TGFRs therapy alone work as sufficiently as the combination (Figure 117). In vitro senescent B16F10 melanoma model

Next, in vitro killing of senescent B16F 10 melanoma cells by activated mouse NK cells was evaluated. B16F10 senescence cells (B16F10-SNC) cells were labelled with CellTrace violet and incubated for 16 hrs with different E:T ratio of in vitro 2t2- activated mouse NK cells (isolated from spleen of C57BL/6 mice injected with

TGFRtl5-TGFRs 10 mg/kg for 4 days). The cells were trypsinized, washed and resuspended in complete media containing propidium iodide (PI) solution. The cytotoxicity was assessed by flow cytometry (Figure 118).

In vivo efficacy of TGFRtl 5-TGFRs in a melanoma mouse model

Next, the in vivo efficacy of TGFRtl 5-TGFRs in a melanoma mouse model was evaluated using a combination treatment containing TGFRtl 5-TGFRs. Figure 119A shows a schematic of the treatment regimen. C57BL/6 mice were subcutaneously injected with 0.5xl0 ⁶ B16F10 melanoma cells, and treated with three doses of chemotherapy docetaxel (10 mg/kg) at day 1, day 4 and day 7 followed by treatment with a single dose of a combination immunotherapy of TGFRtl 5-TGFRs (3 mg/kg) + TA99 (200 pg) (anti- Trpl antibody). As shown in Figure 119B, tumor growth was monitored by caliper measurement, and tumor volume was calculated using the formula V = (L ^c W ² )/2, where L is the largest tumor diameter and W is the perpendicular tumor diameter (N=10, ****p <0.001, Multiple t test analyses). Next, peripheral blood analysis of immune cell subsets in B16F10 tumor model was carried out. Blood was drawn from submandibular vein pre- immunotherapy and post-immunotherapy treatment at days 2, 5 and 8. The RBCs were lysed in ACK lysis buffer and the lymphocytes were washed and stained with

fluorescently labelled anti-NKl. l, anti-CD8 and anti-CD4 antibodies. The cells were analyzed by Flow Cytometry (Celesta-BD Bioscience).

Example 79: 7tl5-21sl37L (long version) fusion protein creation and

characterization

A fusion protein complex was generated comprising of IL-21/IL- l5RaSu/CDl37L and IL-7/TF/IL-15 fusion proteins. Specifically, a construct was made linking the IL-7 sequence to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15. The nucleic acid and protein sequences of a construct comprising IL-7 linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the 7tl 5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AG AT C AAGACCTGCTGGAAC AAGATCCTGATGGGC ACC AAGGAGC AT

(Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7tl 5 fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL7) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A AL GE AQPTK SLEENK SLKEQKKLNDLCFLKRLLQEIKT C WNKILMGTKEH

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS

The nucleic acid and protein sequences of the 2l sl37L are shown below. The nucleic acid sequence of the 2l sl37L construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCC AAGGAGTTCCTGGAGAGGTTC AAGTCCCTGCTGC AGAAGAT GAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(Human IL-15R a sushi domain) ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

(( G4S)3 linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

(Human CD137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGAC CTGCGGC AGGGC AT GTTT GCGC AGCTGGT GGCCC AAAAT GTTCTGCTGATCG ATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGAC GGGGGGC C T G AGC T AC A A AG AGG AC AC G A AGG AGC T GGT GGT GGC C A AGGC TGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCG AGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCT GCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGA GGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCG GCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTG GCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAA ATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence of 2l sl37L fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEW S AF SCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHL S SRTHGSED S

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNKAT NVAHWTTPSLKCIR

((G4S)3 linker) GGGGS GGGGS GGGGS

(Human CD137L)

REGPEL SPDDP AGLLDLRQGMF AQL VAQNVLLIDGPL S W Y SDPGL AGV S LTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSA AGAAAL ALTVDLPP AS SEARNS AFGFQGRLLHLS AGQRLGVHLHTEARARHAW

QLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The IL-2l/IL-l5RaSu/CDl37L and IL-7/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO- Kl cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble IL-7/TF/IL-15: IL-2l/IL-l5RaSu/CDl37L protein complex (referred to as 7tl5-2lsl37L), which can be purified by anti-TF antibody affinity and other chromatography methods. Purification elution chromatograph of 7tl5-21sl37L using anti-TF antibody affinity column

7tl5-2lsl37L (Figures 120 and 121) harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in Figure 122, the anti-TF antibody affinity column bound to 7tl5-2lsl37L which contains TF as a fusion partner. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min. Figure 123 shows the analytical SEC profile of 7tl5-2lsl37L.

Example 80: 7tl5-21sl37L (short version) fusion protein generation and

characterization

A fusion protein complex was generated comprising of IL-21/IL- l5RaSu/CDl37L and IL-7/TF/IL-15 fusion proteins. Specifically, a construct was made linking the IL-7 sequence to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15. The nucleic acid and protein sequences of a construct comprising IL-7 linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of 7tl5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT

GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AG AT C AAGACCTGCTGGAAC AAGATCCTGATGGGC ACC AAGGAGC AT

(Human Tissue Factor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7tl 5 fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A AL GE AQPTK SLEENK SLKEQKKLNDLCFLKRLLQEIKT C WNKILMGTKEH

(Human Tissue Factor 219) S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS

The nucleic acid and protein sequences of the 2l sl37L (short version) are shown below. The nucleic acid sequence of 2l sl37L (short version) construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCC AAGGAGTTCCTGGAGAGGTTC AAGTCCCTGCTGC AGAAGAT GAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG

AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA

AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA

CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

((G4S)3 linker) GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD 137 Ligand short version)

GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTG

GTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACC

CAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACA

CGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTA

GAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGC

TGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACC

GTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCA

GGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCAC

ACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCT

TGGGACTCTTCCGGGTGACCCCCGAAATC

The amino acid sequence of the 2lsl37L (short version) construct (including signal peptide sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEW S AF SCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHL S SRTHGSED S

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNKAT NVAHWTTPSLKCIR

((G4S)3 linker)

GGGGS GGGGS GGGGS

(Human CD 137 Ligand short version)

DP AGLLDLRQGMF AQL VAQNVLLIDGPLS W Y SDPGL AGV SLT GGLS YKE DTKEL VVAKAGVYYVFF QLELRRVVAGEGSGS V SL ALHLQPLRS AAGAAAL AL T VDLPP AS SEARN S AF GFQGRLLHL S AGQRLGVHLHTE ARARHAW QLTQGAT V LGLFRVTPEI The IL-2l/IL-l5RaSu/CDl37L (short version) and IL-7/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble IL-7/TF/IL-15: IL-21/IL- l5RaSu/CDl37L protein complex (referred to as 7tl5-2lsl37L (short version)), which can be purified by anti-TF antibody affinity and other chromatography methods.

Binding of 7tl5-21sl37L (short version) to CD 137 (4. IBB)

On day 1, a 96-well plate was coated with 100 pL (2.5 pg/mL) of GAH IgG Fc (G-102-C, R&D Systems) in R5 (coating buffer) or R5 only and incubated at 4°C, overnight. On day 2, the plates were washed three times and blocked with 300 pL of 1% BSA in PBS at 37°C for 2 hrs. 10 ng/mL of 4.lBB/Fc (838-4B, R&D Systems) was added at 100 pL/well and incubated for 2 hrs at RT. After three washes, the 7tl 5- 2lsl37L or 7tl5-2ls serially diluted at a 1/3 ratio (starting at 10 nM), and incubated at 4°C overnight. On day 3, following 3 washes, 300 ng/mL of biotinylated-anti-hTF antibody (BAF2339, R&D Systems) was added at 100 pL per well and incubated at RT for 2 hrs. The plate was then washed three times and incubated with 0.25 pg/mL of HRP- SA (Jackson ImmuneResearch) at 100 pL per well for 30 min, followed by 3 washes and incubation with 100 pL of ABTS for 2 mins at RT. Absorbance was read at 405 nm. As shown in Figure 124, 7tl5-2lsl37L (short version) showed significant interaction with 4. lBB/Fc (blue line) as compared to 7tl5-2ls.

Detection ofIL-15, IL-21, andlL-7 in 7tl5-21sl37L (short version) with ELISA A 96-well plate was coated with 100 pL (8 pg/mL) of anti-TF antibody in R5 (coating buffer) and incubated at RT for 2 hrs. The plates were washed 3 times and blocked with 100 pL of 1% BSA in PBS. 7tl5-2lsl37L (short version), serially diluted at a 1 :3 ratio was added, and incubated at RT for 60 min. After three washes, 50 ng/mL of biotinylated-anti-ILl5 antibody (BAM247, R&D Systems), 500 ng/mL of biotinylated-anti-IL2l antibody (13-7218-81, R&D Systems), or 500 ng/mL of biotinylated-anti-IL7 antibody (506602, R&D Systems) was added to the wells and incubated at RT for 60 min. After three washes and incubation with 0.25 gg/mL of HRP- SA (Jackson ImmunoResearch) at 100 pL per well was carried out for 30 min at RT, followed by four washes and incubation with 100 gL of ABTS for 2 mins at RT.

Absorbance was read at 405 nm. As shown in Figures 125A-125C, the IL-15, IL-21, and IL-7 domains in 7tl5-2lsl37L (short version) were detected by the respective antibodies.

The IL-15 in 7tl5-lsl37L (short version) promotes IL2R /fiy containing CTLL2 cell proliferation

To evaluate the IL-15 activity of 7tl5-2lsl37L (short version), 7tl5-2lsl37L (short version) was compared with recombinant IL15 in promoting proliferation of IL2Ra ?y expressing CTLL2 cells. IL- 15 -dependent CTLL2 cells were washed 5 times with IMDM-l0% FBS and seeded to the wells at 2 x 10 ⁴ cells/well. Serially diluted 7tl 5- 2lsl37L (short version) or IL-15 were added to the cells (Figure 126). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by adding 10 pL of WST1 to each well on day 3 and incubated for an additional 3 hours in a CO2 incubator at 37°C. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in Figure 126, 7tl5-2lsl37L (short version) and IL-15 promoted CTLL2 cell proliferation. The EC50 of 7tl5-2lsl37L (short version) and IL-15 was 55.91 pM and 6.22 pM. respectively.

The IL-21 in 7tl5-lsl37L (short version) promotes IL21R containing B9 cell proliferation

To evaluate the IL-21 activity of 7tl5-2lsl37L (short version), 7tl5-2lsl37L

(short version) was compared with recombinant IL-21 in promoting proliferation of IL- 21R expressing B9 cells. IL-21R containing B9 cells were washed 5 times with RPMI- 10% FBS and seeded to the wells at 1 x 10 ⁴ cells/well. Serially diluted 7tl5-2lsl37L (short version) or IL-21 were added to the cells (Figure 127). Cells were incubated in a CO2 incubator at 37°C for 5 days. Cell proliferation was detected by adding 10 pL of WST1 to each well on day 5 and incubated for an additional 4 hours in a CO2 incubator at 37°C. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in Figure 127, 7tl5-2lsl37L (short version) and IL-21 promoted B9 cell proliferation. The ECso of 7tl5-2lsl37L (short version) and IL-21 was 104.1 nM and 72.55 nM. respectively. Example 86: 7tl5-TGFRs fusion protein generation and characterization

A fusion protein complex was generated comprising of TGF Receptor II/IL- l5RaSu and IL-7/TF/IL-15 fusion proteins (Figures 128 and 129). The human TGF Receptor II (Ile24-Aspl59), tissue factor 219, IL-15, and IL-7 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking the IL-7 sequence to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15. The nucleic acid and protein sequences of a construct comprising IL-7 linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the 7tl 5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT

GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AG AT C AAGACCTGCTGGAAC AAGATCCTGATGGGC ACC AAGGAGC AT

(Human Tissue Factor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7tl 5 fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A AL GE AQPTK SLEENK SLKEQKKLNDLCFLKRLLQEIKT C WNKILMGTKEH

(Human Tissue Factor 219) S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS

Constructs were also made by attaching two TGF Receptor II directly to the IL- l 5RaSu chain which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the TGF Receptor II linked to the N-terminus of IL-l 5RaSu are shown below.

The nucleic acid sequence of the TGFRs construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human ΊCiIb Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGT GC AGA AGAGCGT GAAT AATGAC AT GATCGTGACCGAT AAC AATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG

CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC

TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA

AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA

GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT

TTAGCGAGGAATACAATACCAGCAACCCCGAC

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG

AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA

AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA

CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence of TGFRs fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human TGhft Receptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLET V CHDPKLP YHDFILED A A SPKCIMKEKKKPGETFFMC S C S SDE CNDNIIF SEEYNT SNPD

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNK AT NVAHWTTPSLKCIR

Effect of 7tl5-TGFRs on TGFfl activity in HEK-Blue TGFf cells

To evaluate the activity of TGFpR in 7tl5-TGFRs, the effect of 7tl5-TGFRs on the activity of TGFpl in HEK-Blue TGFp cells was analyzed. HEK-Blue TGFp cells (Invivogen) were washed twice with pre-warmed PBS and resuspended in the testing medium (DMEM, 10% heat-inactivated FCS, lx glutamine, lx anti-anti, and 2x glutamine) at 5 x 10 ⁵ cells/mL. In a flat-bottom 96-well plate, 50 pL cells were added to each well (2.5 x 10 ⁴ cells/well) and followed with 50 pL O.lnM TGFp i (R&D systems). 7tl5-TGFRs or TGFR-Fc (R&D Systems) prepared at al :3 serial dilution was then added to the plate to reach a total volume of 200 pL. After 24hrs of incubation at 37°C, 40 pL of induced HEK-Blue TGFp cell supernatant was added to 160 pL pre-warmed

QUANTI-Blue (Invivogen) in a flat-bottom 96-well plate, and incubated at 37°C for 1-3 hrs. The OD values were then determined using a plate reader (Multiscan Sky) at 620-655 nM. See, Figure 130. The ICso of each protein sample was calculated with GraphPad Prism 7.04. The ICso of 7tl5-TGFRs and TGFR-Fc were 1142 pM and 558.6 pM respectively. These results showed that the TGFpR in 7tl5-TGFRs was able to block the activity of TGFpl in HEK-Blue TGFP cells.

Detection of IL-15, TGIflRII, andIL-7 in 7tl5-TGFRs with ELISA

A 96-well plate was coated with 100 pL (8 pg/mL) of anti-TF antibody in R5

(coating buffer) and incubated at room temperature (RT) for 2 hrs. The plates were washed three times and blocked with 100 pL of 1% BSA in PBS. Serial dilution of 7tl 5- TGFRs (1 :3 ratio) was added, and incubated at RT for 60 mins. After 3 washes, 50 ng/mL of biotinylated-anti-IL-l5 antibody (BAM247, R&D Systems), 200 ng/mL of biotinylated-anti-TGFbRII antibody (BAF241, R&D Systems), or 500 ng/mL of biotinylated-anti-IL-7 antibody (506602, R&D Systems) was added and incubated at RT for 60 min. Following three washes, incubation with 0.25 pg/mL of HRP-SA (Jackson ImmunoResearch) at 100 pL per well was carried out for 30 min at RT, followed by 4 washes and incubation with 100 pL of ABTS for 2 mins at RT. Absorbance was read at 405 nm. As shown in Figures 131A-131C, the IL-15, TGFR, and IL-7 in 7tl5-TGFRs were detected by the respective antibodies.

The IL-15 in 7tl5-TGFRs promotes IL-2Rb and common g chain containing 32Όb cell proliferation

To evaluate the activity of IL-15 in 7tl5-TGFRs, 7tl5-TGFRs was compared to recombinant IL-15 using 32ϋb cells that express IL2RP and common g chain, and evaluating their effects on promoting cell proliferation. IL-15 dependent 32ϋb cells were washed 5 times with IMDM-lO% FBS and seeded in the wells at 2 x 10 ⁴ cells/well. Serially-diluted 7tl5-TGFRs or IL-15 were added to the cells (Figure 132). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by adding 10 pL of WST1 to each well on day 3 and incubating for an additional 3 hours in a CO2 incubator at 37°C. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in Figure 132, 7tl5-TGFRs and IL-15 promoted 32ϋb cell proliferation, with the EC50 of 7tl5-TGFRs and IL-15 being 126 nM and 16.63 pM, respectively.

Purification elution chromatograph of 7tl5-TGFRs using anti-TF antibody affinity column

7tl5-TGFRs harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in Figure 133, the anti-TF antibody affinity column can bind 7tl 5- TGFRs which contains TF as a fusion partner of 7tl5-TGFRs. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Reduced SDS-PAGE analysis of 7tl5-TGFRs

To determine the purity and molecular weight of the protein, 7tl5-TGFRs protein sample purified with anti-TF antibody affinity column was analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) method under reduced condition. After electrophoresis, the gel was stained with

InstantBlue for about 30 min, followed by destaining overnight in purified water. To verify that the 7tl5-TGFRs protein undergoes glycosylation after translation in CHO cells, a deglycosylation experiment was conducted using the Protein Deglycosylation Mix II kit from New England Biolabs and the manufacturer’s instructions. Figure 134 shows reduced SDS-PAGE analysis of the sample in non-deglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state. These results showed that the protein is glycosylated when it is expressed in CHO cells. After deglycosylation, the purified sample showed expected molecular weights (55 kDa and 39 kDa) in reduced SDS gel. Lane M was loaded with 10 ul of SeeBlue Plus2 Prestained Standard.

Characterization of 7tl5-TGFRs

7tl5-TGFRs is a multi-chain polypeptide (a type A multi-chain polypeptide described herein) that includes the first polypeptide that is a soluble fusion of human IL-7, human tissue factor 219 fragment and human IL- 15 (7tl 5), and the second polypeptide that is a soluble fusion of single chain two TGFpRII domains and sushi domain of human IL- 15 receptor alpha chain (TGFRs).

CHO cells were co-transfected with 7tl 5 and TGFRs vectors. The 7tl5-TGFRs complex was purified from the transfected CHO cell culture supernatant. The IL-7, IL-15, TGFp receptor and tissue factor (TF) components were demonstrated in the complex by ELISA as shown in Figure 135. A humanized anti-TF monoclonal antibody was used as the capture antibody to determine TF in 7tl5-TGFRs, and biotinylated antibodies against human IL-15 antibody (R&D systems), human IL-7 (Biolegend), anti-TGFp receptor (R&D Systems) were used as the detection antibodies to respectively determine IL-7, IL- 15 and TGFp receptor in 7tl5-TGFRs. Peroxidase conjugated streptavidin (Jackson

ImmunoResearch Lab) and ABTS substrate (Surmodics IVD, Inc.) were then used to detect the bound biotinylated antibodies. The results were analyzed by ELISA (Figure 135).

In vivo characterization of 7tl5-TGFRs in C57BL/6 mice

To determine the immunostimulatory activity of 7tl5-TGFRs in vivo, C57BL/6 mice were subcutaneously treated with control solution (PBS) or 7tl5-TGFRs at 0.3, 1, 3 and 10 mg/kg. The treated mice were euthanized. The mouse spleens were collected and weighed day 4 post treatment. Single splenocyte suspensions were prepared and stained with fluorochrome-conjugated anti-CD4, anti-CD8, and anti-NKl. l antibodies and the percentage of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells was analyzed by flow cytometry. The results showed that 7tl5-TGFRs was effective at expanding splenocytes based on spleen weight (Figure 136A), especially at 1-10 mg/kg. The percentages of CD8 ⁺ T cells and NK cells were higher compared to control-treated mice (Figure 136B) at all doses tested.

CD44 Expression of CD4 ⁺ and CDS T cells

It has been known that IL-15 induces CD44 expression on T cells and

development of memory T cells. CD44 expression of CD4 ⁺ and CD8 ⁺ T cells in the 7tl 5- TGFRs treated mice were assessed. C57BL/6 mice were subcutaneously treated with 7tl5-TGFRs. The splenocytes were stained with fluorochrome-conjugated anti-CD4, anti-CD8 and anti-CD44 monoclonal antibodies for immunocyte subsets. The

percentages of CD4 ⁺ CD44 ^high T cells of total CD4+ T cells and CD8 ⁺ CD44 ^high T cells of total CD8 ⁺ T cells were analyzed by flow cytometry. As shown in Figures 137A and 137B, 7tl5-TGFRs significantly activated CD4 ⁺ and CD8 ⁺ T cells to differentiate into memory T cells.

Furthermore, the dynamic proliferation of immune cells based on Ki67 expression of splenocytes and cytotoxicity potential based on granzyme B expression of the splenocytes induced by 7tl5-TGFRs after the single dose treatment of mouse were also evaluated. C57BL/6 mice were subcutaneously treated with 7tl5-TGFRs at 3 mg/kg. The treated mice were euthanized and the splenocytes were prepared. The prepared splenocytes were stained with fluorochrome-conjugated anti-CD4, anti-CD8, and anti- NK1.1 (NK) antibodies for immunocyte subsets and then intracellularly stained with anti- Ki67 antibody for cell proliferation and anti-granzyme B antibody for cytotoxic marker. The mean fluorescent intensity (MFI) of Ki67 and granzyme B of corresponding immunocyte subsets was analyzed by flow cytometry. As shown in Figures 138A and 138B, in the spleens of mice treated with 7tl5-TGFRs, the expression of Ki67 and granzyme B by CD8 ⁺ T cells and NK cells increased compared with PBS control treatment. These results demonstrate that 7tl5-TGFRs is not only to increase numbers of CD8 ⁺ T cells and NK cells but also enhance potential cytotoxicity of these cells.

Additionally, cytotoxicity of the mouse splenocytes against tumor cells was also evaluated. Mouse Yac-l cells were labeled with CellTrace Violet and used as tumor target cells. The splenocytes were prepared from 7tl5-TGFRs-treated mice and used as effector cells. The target cells were mixed with effector cells at E:T ratio = 10: 1 in RPMI-10 medium with or without 7tl5-TGFRs at 100 nM and incubated at 37°C for 20 hours. Target Yac-l cell inhibition was assessed by analysis of viable violet-labeled Yac-l cells using flow cytometry. Percentage of Yac-l inhibition was calculated using a formula, (1 -viable Yac-l cell number in experimental sample/viable Yac-l cell number in the sample without splenocytes) x 100. As shown in Figure 139, 7tl5-TGFRs-treated mouse splenocytes had stronger cytotoxicity against Yac-l cells than the control mouse splenocytes and addition of 7tl5-TGFRs during cytotoxic assay further enhanced cytotoxicity of splenocytes against Yac-l target cells. Example 81: TGFRtl5-21sl37L fusion protein generation and characterization

A fusion protein complex was generated comprising IL-2l/IL-l5RaSu/CDl37L and TGFp Receptor II/TF/IL- 15 fusion proteins. The human TGFP Receptor II (II 624- Asp 159), tissue factor 219, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking two TGFp Receptor II sequences with a G4S(3) linker to generate a single chain version of TGFP Receptor II and then directly linking to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15.

The nucleic acid sequence of the TGFRtl5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human ΊC Ib Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC

CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT

GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC

ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA

GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT

AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG

GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA

CGT GC AGA AGAGCGT GAAT AATGAC AT GATCGTGACCGAT AAC AATGGCGCC

GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA

CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG

CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC

TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA

AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA

GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT

TTAGCGAGGAATACAATACCAGCAACCCCGAC

(Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Human IL-15) AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRtl 5 fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human KIIb Receptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLET V CHDPKLP YHDFILED A A SPKCIMKEKKKPGETFFMC S C S SDE CNDNIIF SEEYNT SNPD

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS The nucleic acid and protein sequences of the 2l sl37L are shown below. The nucleic acid sequence of the 2l sl37L construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCC AAGGAGTTCCTGGAGAGGTTC AAGTCCCTGCTGC AGAAGAT GAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG

AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA

AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA

CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

((G4S)3 linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

(Human CD137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGAC CTGCGGC AGGGC AT GTTT GCGC AGCTGGT GGCCC AAAAT GTTCTGCTGATCG ATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGAC GGGGGGC C T G AGC T AC A A AG AGG AC AC G A AGG AGC T GGT GGT GGC C A AGGC TGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCG AGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCT GCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGA GGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCG GCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTG

GCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAA

ATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA The amino acid sequence of 2lsl37L fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWS AF SCF

QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHL S SRTHGSED S

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNKAT NVAHWTTPSLKCIR

((G4S)3 linker)

GGGGS GGGGS GGGGS

(Human CD137L)

REGPEL SPDDP AGLLDLRQGMF AQL VAQNVLLIDGPL S W Y SDPGL AGV S LTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSA AGAAAL ALTVDLPP AS SEARN S AF GF QGRLLHLS AGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The IL-2l/IL-l5RaSu/CDl37L and TGFR/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO- Kl cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble TGFR/TF/IL-15: IL-2l/IL-l5RaSu/CDl37L protein complex (referred to as TGFRtl5-2lsl37L), which can be purified by anti-TF antibody affinity and other chromatography methods. See, Figures 140 and 141. Purification elution chromatograph of TGFRtl5-21sl37L using anti-TF antibody affinity column

TGFRtl5-2lsl37L harvest from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in Figure 142, the anti-TF antibody affinity column bound to TGFRtl5- 2lsl37L which contains TF as a fusion partner of TGFRtl5-2lsl37L. The buffer- exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Example 82: TGFRtl5-TGFRs21 fusion protein generation and characterization

A fusion protein complex was generated comprising of TGF Receptor II/IL- l5RaSu/IL-2l and TGF Receptor II/TF/IL-15 fusion proteins. The human TGF

Receptor II (Ile24-Aspl59), tissue factor 219, IL-21, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking two TGF Receptor II sequences with a G4S(3) linker to generate a single chain version of TGF Receptor II and then directly linking to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15. The nucleic acid and protein sequences of a construct comprising two TGF Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the TGFRU5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human ΊC Ib Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGT GC AGA AGAGCGT GAAT AATGAC AT GATCGTGACCGAT AAC AATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC

(Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG

CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT

TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT

ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC

ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA

CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC

ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC

AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT

GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA

AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC

AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT

T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRtl5 fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human KIIb Receptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLET V CHDPKLP YHDFILED A A SPKCIMKEKKKPGETFFMC S C S SDE CNDNIIF SEEYNT SNPD

(Human Tissue Factor 219) S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS

Constructs were also made by attaching two TGF Receptor II directly to the IL- l 5RaSu chain, followed by the N-terminus coding region of IL-21, which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the TGF Receptor II linked to the N-terminus of IL-l 5RaSu following with the N-terminus of IL-21 are shown below.

The nucleic acid sequence of the TGFRs2l construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human ΊCiIb Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGT GC AGA AGAGCGT GAAT AATGAC AT GATCGTGACCGAT AAC AATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

(Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCC AAGGAGTTCCTGGAGAGGTTC AAGTCCCTGCTGC AGAAGAT GAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence of TGFRs2l fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human KIIb Receptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLET V CHDPKLP YHDFILED A A SPKCIMKEKKKPGETFFMC S C S SDE CNDNIIF SEEYNT SNPD

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNKAT NVAHWTTPSLKCIR

(Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEW S AF SCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHL S SRTHGSED S

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The TGFR/IL-l5RaSu/IL-2l and TGFR/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO- Kl cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble TGFR/TF/IL-l5:TGFR/IL-l5RaSu/IL-2l protein complex (referred to as TGFRtl5-TGFRs2l), which can be purified by anti-TF antibody affinity and other chromatography methods. See, Figures 143 and 144.

Purification elution chromatograph of TGFRtl5-TGFRs21 using anti-TF antibody affinity column

TGFRtl5-TGFRs2l harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in Figure 145, the anti-TF antibody affinity column bound to TGFRtl5-TGFRs2l which contains TF as a fusion partner. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Reduced SDS-PAGE analysis of TGFRtl 5-TGFRs21

To determine the purity and molecular weight of the protein, TGFRtl 5 -TGFRs2l protein sample purified with anti-TF antibody affinity column was analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS- PAGE) method under reduced condition. After electrophoresis, the gel was stained with InstantBlue for about 30 min, followed by destaining overnight in purified water.

To verify that the TGFRtl 5 -TGFRs2l protein undergoes glycosylation after translation in CHO cells, a deglycosylation experiment was conducted using the Protein Deglycosylation Mix II kit from New England Biolabs and the manufacturer’s instructions. Figure 146 shows the reduced SDS-PAGE analysis of the sample in non- deglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state. It is clear that the protein is glycosylated when it is expressed in CHO cells. After deglycosylation, the purified sample showed expected molecular weights (69 kDa and 55 kDa) in reduced SDS gel. Lane M was loaded with 10 ul of SeeBlue Plus2 Prestained Standard. Immunostimulation of TGFRtl 5-TGFRs21 in C57BL/6 mice

TGFRtl 5-TGFRs2l is a multi-chain polypeptide (a type A multi-chain polypeptide described herein) that includes the first polypeptide that is a soluble fusion of single chain two TGFpRII domains, human tissue factor 219 fragment and human IL-15 (TGFRtl 5), and the second polypeptide that is a soluble fusion of single chain two TGFpRII domains, sushi domain of human IL-15 receptor alpha chain and human IL-21 (TGFRs2l). CHO cells were co-transfected with TGFRtl 5 and TGFRs2l vectors. The TGFRtl5-TGFRs2l complex was purified from the transfected CHO cell culture supernatant. The TGFP receptor, IL-15, IL-21 and tissue factor (TF) components were demonstrated in the complex by ELISA as shown in Figures 147 A and 147B. A humanized anti-TF monoclonal antibody was used as the capture antibody to determine TF in TGFRtl5-TGFRs2l, biotinylated anti-human IL-15 antibody (R&D systems), biotinylated anti-human TGFP receptor antibody (R&D systems, and biotinylated anti human IL-21 antibody (R&D Systems) were used as the detection antibodies to respectively determine IL-15, TGFP receptor, and IL-21 in TGFRtl5-TGFRs2l. For detection, peroxidase conjugated streptavidin (Jackson ImmunoResearch Lab) and ABTS were used.

Wild type C57BL/6 mice were treated subcutaneously with either control solution (PBS) or with TGFRtl5-TGFRs2l at 3 mg/kg. Four days after treatment, spleen weight and the percentages of various immune cell types present in the spleen were evaluated. As shown in Figure 148A, the percentages of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells present in the spleen of control -treated and TGFRtl 5 -TGFRs2l -treated mice were evaluated. The dynamic proliferation of immune cells based on Ki67 expression after TGFRtl 5 -TGFRs2l treatment was also evaluated. The splenocytes were stained with fluorochrome-conjugated anti-CD4, anti-CD8, and anti-NKl.l (NK) antibodies and then intracellularly stained with anti-Ki67 antibody. The percentage of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells and the mean fluorescent intensity (MFI) of Ki67 of corresponding immunocyte subsets were analyzed by flow cytometry (Figures 148A and 148B).

Furthermore, cytotoxicity potential based on granzyme B expression of the splenocytes induced by TGFRtl 5-TGFRs2l after the single dose treatment of mouse was also evaluated. As shown in Figure 149, in the spleens of mice treated with TGFRtl 5- TGFRs2l, the expression of granzyme B by NK cells increased after treatment. The splenocytes from TGFRtl 5-TGFRs2l -treated mice were stained with fluorochrome- conjugated anti-CD4, anti-CD8, and anti-NKl. l (NK) antibodies and then intracellularly stained with anti-granzyme B antibody. The mean fluorescent intensity (MFI) of granzyme B of corresponding immunocyte subsets was analyzed by flow cytometry (Figure 149). As shown in Figure 148A, in the spleens of mice treated with TGFRtl5- TGFRs2l, the percentages of CD8 ⁺ T cells and NK cells both increased on day 4 after a single TGFRtl5-TGFRs2l treatment. These results demonstrate that TGFRtl5- TGFRs2l is able to induce immune cells to proliferate in mouse spleen, in particular CD8 ⁺ T cells and NK cells.

Additionally, cytotoxicity of the mouse splenocytes against tumor cells was also evaluated. Mouse Yac-l cells were labeled with CellTrace Violet and used as tumor target cells. The splenocytes were prepared from TGFRtl5-TGFRs2l -treated mice and used as effector cells. The target cells were mixed with effector cells at E:T ratio= 10: 1 in RPMI-10 medium with or without TGFRtl5-TGFRs2l at 100 nM and incubated at 37°C for 24 hours. Target Yac-l cell inhibition was assessed by analysis of viable violet- labeled Yac-l cells using flow cytometry. Percentage of Yac-l inhibition was calculated using a formula, (1 -[viable Yac-l cell number in experimental sample]/[viable Yac-l cell number in the sample without splenocytes]) x 100. As shown in Figure 150, TGFRU5- TGFRs2l -treated mouse splenocytes had stronger cytotoxicity against Yac-l cells than the control mouse cells in the presence of TGFRtl5-TGFRs2l during cytotoxic assay (shown in legend of Figure 150).

Example 83: TGFRtl5-TGFRsl6 fusion protein generation

A fusion protein complex was generated comprising of TGF Receptor II/IL- l5RaSu/ anti-CDl6scFv and TGF Receptor II/TF/IL-15 fusion proteins (See, Figures 151 and 152). The human TGF Receptor II (Ile24-Aspl59), tissue factor 219, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking two TGF Receptor II sequences with a G4S(3) linker to generate a single chain version of TGF Receptor II and then directly linking to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising two TGF Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below. The nucleic acid sequence of the TGFRU5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human ΊC Ib Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGT GC AGA AGAGCGT GAAT AATGAC AT GATCGTGACCGAT AAC AATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC

(Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG

CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT

TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT

ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA

ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC

ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA

CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC

AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT

GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA

AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC

AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT

T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRtl5 fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human KIIb Receptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLET V CHDPKLP YHDFILED A A SPKCIMKEKKKPGETFFMC S C S SDE CNDNIIF SEEYNT SNPD

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS

Constructs were also made by attaching two TGF Receptor II directly to the IL- l 5RaSu chain, followed by the anti-CD l6scFv sequence, which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the TGF Receptor II linked to the N-terminus of IL-l 5RaSu following with the anti-CD l6scFv sequence are shown below.

The nucleic acid sequence of the TGFRsl6 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human TGF b Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGT GC AGA AGAGCGT GAAT AATGAC AT GATCGTGACCGAT AAC AATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

(Anti-human CD scFv)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACC GTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGT ACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAA CAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCG CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTG CAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAG CTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGA GG AGG AT C C G AGGT GC AGC T GGT GG AGT C C GG AGG AGG AGT GGT G AGGC C T GGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTA CGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCC GGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCA GGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAA CTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCC CTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG

The amino acid sequence of TGFRsl6 fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human TGF b Receptor II) IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLET V CHDPKLP YHDFILED A A SPKCIMKEKKKPGETFFMC S C S SDE CNDNIIF SEEYNT SNPD

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNKAT NVAHWTTPSLKCIR

(Anti-human CD 16scFv)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRP S GIPDRF S GS S S GNT ASLTIT GAQ AEDE AD Y Y CN SRD S S GNH V VF GGGTKL TV GHGGGGS GGGGS GGGGSE V QL VES GGGVVRPGGSLRL S C A ASGF TFDD Y GM SWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLR AEDTAVYY C ARGRSLLFDYW GQGTLVT V SR

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The TGFR/IL-l5RaSu/anti-CDl6scFv and TGFR/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005; 16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble TGFR/TF/IL-l5:TGFR/IL- l5RaSu/anti-CDl6scFv protein complex (referred to as TGFRtl5-TGFRsl6), which can be purified by anti-TF antibody affinity and other chromatography methods.

Example 84: The TGFRtl5-TGFRsl37L fusion protein generation

A fusion protein complex was generated comprising of TGF Receptor II/IL- !5RaSu/ CD137L and TGF Receptor IETF/IL-15 fusion proteins (see, Figures 153 and 154). The human TGF Receptor II (Ile24-Aspl59), tissue factor 219, CD137L, and IL- 15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking two TGF Receptor II sequences with a G4S(3) linker to generate a single chain version of TGF Receptor II and then directly linking to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising two TGF Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the TGFRtl5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human TGF Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGT GC AGA AGAGCGT GAAT AATGAC AT GATCGTGACCGAT AAC AATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC

(Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRtl5 fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human TGF b Receptor II) IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLET V CHDPKLP YHDFILED A A SPKCIMKEKKKPGETFFMC S C S SDE CNDNIIF SEEYNT SNPD

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS

Constructs were also made by attaching two TGF Receptor II directly to the IL- l 5RaSu chain, followed by a (G4S)3 linker and the CD137L sequence, which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the TGF Receptor II linked to the N-terminus of IL-l 5RaSu following with a (G4S)3 linker and the CD137L sequence are shown below.

The nucleic acid sequence of the TGFRsl37L construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human TGF b Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC

GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA

GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGT GC AGA AGAGCGT GAAT AATGAC AT GATCGTGACCGAT AAC AATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

((G4S)3 linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

(Human CD137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGAC CTGCGGC AGGGC AT GTTT GCGC AGCTGGT GGCCC AAAAT GTTCTGCTGATCG ATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGAC GGGGGGC C T G AGC T AC A A AG AGG AC AC G A AGG AGC T GGT GGT GGC C A AGGC TGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCG AGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCT GCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGA GGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCG GCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTG GCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAA

ATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence of TGFRsl37L fusion protein (including the leader sequence) is as follows:

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human TGF b Receptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLET V CHDPKLP YHDFILED A A SPKCIMKEKKKPGETFFMC S C S SDE CNDNIIF SEEYNT SNPD

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNK AT NVAHWTTPSLKCIR

((G4S)3 linker)

GGGGS GGGGS GGGGS

(Human CD137L)

REGPEL SPDDP AGLLDLRQGMF AQL VAQNVLLIDGPL S W Y SDPGL AGV S LTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSA AGAAAL ALTVDLPP AS SEARN S AF GF QGRLLHLS AGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The TGFR/IL-l5RaSu/CDl37L and TGFR/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005; 16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble TGFR/TF/IL-l5:TGFR/IL-l5RaSu/CDl37L protein complex (referred to as TGFRtl5-TGFRsl37L), which can be purified by anti-TF antibody affinity and other chromatography methods. Example 85. Production and characterization of the Exemplary Single-Chain Chimeric Polypeptide 2t2

An exemplary single-chain chimeric polypeptide including a first target-binding domain that binds to an IL-2 receptor, a soluble human tissue factor domain, and a second target-binding domain that binds to an IL-2 receptor was generated (IL-2/TF/IL-2, or 2t2) (Figure 155). The nucleic acid and amino acid sequences of this single-chain chimeric polypeptide are shown below.

Nucleic Acid Encoding Exemplary Single-Chain Chimeric Polypeptide (IL-2/TF/IL- 2) (SEQ ID NO: 109)

( Signal peptide )

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(First IL-2 fragment)

GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCAT TTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACC CCAAGCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCAC CGAGCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAG GTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAAT CAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTC ATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTT GGATCACCTTCTGCCAGTCCATCATCTCCACTTTAACC

(Human tissue factor 219 form)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA

ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC

ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA

CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC

ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC

AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT

GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA

AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC

AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT

T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Second IL-2 fragment)

GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCAT TT AC T GC T GGATTT AC AGAT GATTTT GA AT GGA ATT A AT A ATT AC A AGA AT C C CAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACA GAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAG TGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAAT C AGC A AT AT C A AC GT A AT AGTT C T GGA AC T A A AGGG AT C T G A A AC A AC AT T C ATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGAT GGATTACCTTTTGTCAAAGCATCATCTCAACACTAACT

Exemplary Single-Chain Chimeric Polypeptide (IL-2/TF/IL-2) (SEQ ID NO: 108)

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-2)

APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKK A TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE Y ADET ATI VEFLNRWITF C Q SII S TLT

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGENY CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-2)

APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKK A TELKHLQCLEEELKPLEEVLNLAQ SKNFHLRPRDLI SNIN VIVLELKGSETTFMCE

Y ADET ATI VEFLNRWITF C Q SII S TLT

The nucleic acid encoding IL-2/TF/IL-2 was cloned into a modified retrovirus expression vector as described previously (Hughes et al., Hum Gene Hier 16:457-72, 2005). The expression vector encoding IL-2/TF/IL-2 was transfected into CHO-K1 cells.

Expression of the expression vector in CHO-K1 cells allowed for secretion of the soluble IL-2/TF/IL-2 single-chain chimeric polypeptide (referred to as 2t2), which can be purified by anti-TF antibody affinity and other chromatography methods. IL-2 and 2t2 promoted IL-2R and common g chain containing 32Όb cell proliferation in a similar manner

To evaluate the IL-2 activity of 2t2, 2t2 was compared with recombinant IL-2 for promoting proliferation of 32ϋb cells that express IL-2R. and common g chain. IL-2 dependent 32ϋb cells were washed 5 times with IMDM-l0% FBS and seeded to the wells at 2 x 10 ⁴ cells/well. Serial dilutions of 2t2 or IL-2 were added to the cells (Figure 156). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by adding 10 mΐ of WST1 to each well on day 3 and incubating for an additional 3 hours in a CO2 incubator at 37°C. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in Figure 156, 2t2 and IL-2 activated 32ϋb cells in a similar manner. The EC50 of 2t2 and IL-2 was 158.1 pM and 140 pM. respectively.

2t2 showed improved ability to promote II -2 R abg containing CTLL-2 cell proliferation as compared to IL-2

To evaluate the IL-2 activity of 2t2, 2t2 was compared with recombinant IL-2 for promoting proliferation of CTLL-2 cells that express IL-2Ra, IL-2R^ and common g chain. IL-2 dependent CTLL-2 cells were washed 5 times with IMDM-lO% FBS and seeded to the wells at 2 x 10 ⁴ cells/well. Serial dilutions of 2t2 or IL-2 were added to the cells (Figure 157). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by adding 10 mΐ of WST1 to each well in the day 3 and incubating for an additional 3 hours in a CO2 incubator at 37°C. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in Figure 157, 2t2 promoted CTLL-2 cell proliferation 4-5-fold stronger than IL-2. The EC50 of 2t2 was 123.2 pM and IL-2 was 548.2 pM. 2t2 suppressed the increase of the high fat-induced hyperglycemia in A pol·; ^~ mice

Six-week-old female ApoE ^/_ mice (Jackson Lab) were fed with standard chow diet or high diet fat containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch (TD88137, Harlan Laboratories) and maintained in the standard conditions. At week 7, mice fed with high fat diet were randomly assigned into the control group and treatment group. Mice then received either 2t2 (treatment group) or PBS (chow diet group and control group) per subcutaneous injection at a dosage of 3 mg/kg. Three days post dosing, the mice were fasted overnight, and blood samples were collected through retro-orbital venous plexus puncture. Overnight fasting glucose levels were measured using a OneTouch Glucometer. As shown in Figure 158, the results showed that 2t2 injection effectively suppresses the increase of glucose levels in ApoE ^/_ mice.

2t2 significantly upregulate the ratio of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T regulatory (Treg) cells in blood lymphocytes

Six-week-old female ApoE ^/_ mice (Jackson Lab) were fed with standard chow diet or high diet fat containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch (TD88137, Harlan Laboratories) and maintained in the standard conditions. At week 7, mice fed with the high fat diet were randomly assigned into control group and treatment group. Mice then received either 2t2 (treatment group) or PBS (chow diet group and control group) per subcutaneous injection at a dosage of 3mg/kg. Three days after the dosing, overnight fasting blood samples were collected through retro-orbital venous plexus puncture and incubated with ACK lysing buffer (Thermo Fisher Scientific) at 37°C for 5 minutes. Samples were then resuspended in FACS buffer (1 X PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) and surface stained with FITC-anti-CD4 and APC-anti-CD25 antibodies (BioLegend) for 30 minutes. Surface-stained samples were further fixed and

premetallized with Fix/Perm buffer (BioLegend) and intracellular stained with PE-anti- Foxp3 antibody (BioLegend). After staining, cells were washed twice with FACs buffer followed by centrifugation at 1500 RPM for 5 minutes at room temperature. The cells were analyzed by flow cytometry (Celesta-BD Bioscience). As shown in Figure 159, 2t2 treatment significantly increased Treg populations in blood lymphocytes (3.5%±0.32) compared to the untreated groups (0.4%±0.l6 for chow diet group and 0.46%±0.09 for high fat diet group).

Purification elution chromatograph of 2t2 from anti-TF antibody affinity column

2t2 harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0.1M acetic acid, pH 2.9. A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 kDa molecular weight cutoff. As shown in Figure 160, the anti-TF antibody affinity column bound to 2t2 which contains TF as a fusion domain. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine, pH 2.5. The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min. Analytical size exclusion chromatography (SEC) analysis of 2t2

To analyze 2t2 using analytical size exclusion chromatography (SEC), a Superdex 200 Increase 10/300 GL gel filtration column (from GE Healthcare) was connected to an AKTA Avant system (from GE Healthcare). The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. A sample containing 2t2 in PBS was injected into the Superdex 200 column using a capillary loop, and analyzed by SEC. The SEC chromatograph of the sample is shown in Figure 161. The SEC results indicated two protein peaks for 2t2. Reduced SDS-PA GE of 2t2

To determine the purity and molecular weight of the protein, 2t2 protein sample purified with anti-TF antibody affinity column was analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) method under reduced condition. After electrophoresis, the gel was stained with InstantBlue for about 30 min, followed by destaining overnight in purified water.

To verify that the 2t2 protein undergoes glycosylation after translation in CHO cells, a deglycosylation experiment was conducted using the Protein Deglycosylation Mix II kit from New England Biolabs according to the manufacturer’s instructions. Figures 162A and 162B show the reduced SDS-PAGE analysis of the sample in non- deglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state. The results show that the 2t2 protein is glycosylated when expressed in CHO cells. After deglycosylation, the purified sample ran with expected molecular weights (56 kDa) in reduced SDS gel. Lane M was loaded with 10 pL of SeeBlue Plus2 Prestained Standard. In vivo characterization of 2t2

2t2 was subcutaneously injected into C57BL/6 mice at various doses to determine the immunostimulatory activity of 2t2 in vivo. Mice were subcutaneously treated with control solution (PBS) or 2t2 at 0.1, 0.4, 2 and 10 mg/kg. The treated mice were euthanized day 3 post treatment. The mouse spleens were collected and weighed day 3 post treatment. Single splenocyte suspensions were prepared, and the prepared splenocytes were stained for CD4 ⁺ T cells, CD8 ⁺ T cells and NK cells (with fluorochrome-conjugated anti-CD4, -CD8, and -NK1.1 antibodies), and analyzed by flow cytometry. The results showed that 2t2 was effective at expanding splenocytes based on spleen weight (Figure 163 A) especially at 0.1-10 mg/kg. The percentage of CD8 ⁺ T cells were higher compared to control -treated mice (Figure 163B) at 2 and 10 mg/kg. The percentage of NK cells were higher compared to control-treated mice (Figure 163B) at all doses tested.

It has been known that IL-2 upregulates CD25 expression by immunocytes. We therefore accessed CD25 expression of CD4 ⁺ T cells, CD8 ⁺ T cells and NK cells in the 2t2 treated mice. C57BL/6 mice were subcutaneously treated with 2t2 as described in the paragraph above. The splenocytes were stained with fluorochrome-conjugated anti-CD4, -CD8, -CD25 and -NK1.1 monoclonal antibodies. The CD25 expression (MFI) of splenocyte subsets was analyzed by flow cytometry. As shown in Figure 164, at the doses and time points tested, 2t2 significantly upregulated CD25 expression by CD4 ⁺ T cells but not CD8+ T cells or NK cells.

The pharmacokinetics of 2t2 in C57BL/6 mice were also investigated. 2t2 was subcutaneously injected into C57BL/6 mice at 1 mg/kg. The mouse blood was drawn from tail vein at various time points as shown in Figure 165 and the serum was prepared. 2t2 concentrations were determined with ELISA (Capture: anti-tissue factor antibody; Detection: biotinylated anti-human IL-2 antibody followed by SA-HRP and ABTS substrate). The half-life of 2t2 was 1.83 hours calculated with PK Solutions 2.0 (Summit Research Services).

2t2 attenuated the formation of high fat-induced atherosclerotic plaques in Apol· ^' mice Six-week-old female ApoE ^/_ mice (The Jackson Laboratory) were fed with standard chow diet or high diet fat (21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch) (TD88137, Harlan Laboratories) and maintained in the standard conditions. At week 7, mice fed with high fat diet (HFD) were randomly assigned into control group and treatment group. Mice were then administrated either 2t2 (treatment group) or PBS (chow diet group and control group) subcutaneously at a dosage of 3mg/kg weekly for 4 weeks. At week 12, all mice were euthanized by isoflurane. Aortas were collected, opened longitudinally and stained with Sudan IV solution (0.5%) using en face method. The percentage of plaque area (red color as shown in Figure 166A) relative to total aorta area was then quantified with Image J software. Figure 166A shows a representative view of atherosclerotic plaques from each group. Figure 166B shows the results of quantitative analysis of atherosclerotic plaques of each group. The percentage of plaque areas in control group (HF Diet) was much higher than the treatment group (HFD+2t2), being 10.28% vs 4.68 %.

2t2 suppresses the progression of type 2 diabetes.

Male BKS.Cg-Dock7 ^m +/+ Lepr^/J (db/db (Jackson Lab)) mice were fed with standard chow diet and received drinking water ad libitum. At the age of six weeks, mice were randomly assigned into control group and treatment group. The treatment group received 2t2 by subcutaneous injection at 3 mg/kg bi-weekly, while control group received vehicle (PBS) only. Overnight fasting glucose levels were measure weekly using a OneTouch Glucometer. The results showed that 2t2 effectively suppressed the increase of glucose levels in BKS.Cg-Dock7 ^m +/+ Lepr ^db /J mice (Figure 167).

2t2 significantly upregulates the ratio of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T regulatory cells in blood lymphocytes after the first injection

Male BKS.Cg-Dock7 ^m +/+ Lepr ^db /J (db/db) (The Jackson Laboratory) mice were fed with standard chow diet and received drinking water ad libitum. At the age of six weeks, mice were randomly assigned into control group and treatment group. The treatment group received 2t2 by subcutaneous injection at 3 mg/kg bi-weekly, while the control group received vehicle (PBS) only. Four days after the first drug injection, overnight fasting blood samples were collected and incubated with ACK lysing buffer (Thermo Fisher Scientific) at 37°C for 5 minutes. Samples were then resuspended in FACS buffer (IX PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) and surface stained with FITC-anti-CD4 and APC-anti-CD25 antibodies (BioLegend) for 30 minutes. Surface-stained samples were further fixed and

premetallized with Fix/Perm buffer (BioLegend) and intracellular stained with PE-anti- Foxp3 antibody (BioLegend). After staining, cells were washed twice with FACs buffer and were analyzed by flow cytometry (Celesta-BD Bioscience). The percentage of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs in blood lymphocytes were measured. As shown in Figure 168, the results showed that 2t2 significantly upregulated the ratio of Tregs in blood lymphocytes. * p<0.05.

Example 86. Production and characterization of the Exemplary Single-Chain Chimeric Polypeptide 15tl5

A second exemplary single-chain chimeric polypeptide including a first target- binding domain that binds to an IL-15 receptor, a soluble human tissue factor domain, and a second target-binding domain that binds to an IL-15 receptor was generated (IL- 15/TF/IL-15 or 15tl 5) (Figure 169). The nucleic acid and amino acid sequences of this single-chain chimeric polypeptide are shown below.

Nucleic Acid Encoding Exemplary Single-Chain Chimeric Polypeptide (IL- 15/TF/IL-15) (SEQ ID NO: 115)

(Signal peptide)

ATGAAGTGGGTGACCTTC ATC AGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(First IL-15 fragment)

AACTGGGT GAACGT GAT C AGCGATTT AAAGAAGATCGAGGATTT AATC CAGAGCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTA GCTGCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCT TTAGAGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTT TAGCCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCA AGGAGT GCGAGGAGCTGGAGGAGAAGAAC AT C A AGGAGTTTTT AC AGAGCT TCGT GC AC ATCGT GC AGAT GTTC AT C AAC ACT AGC

(Human tissue factor 219 form)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA

CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC

ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC

AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT

GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA

AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC

AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT

T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Second IL-15 fragment)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

Exemplary Single-Chain Chimeric Polypeptide (IL-15/TF/IL-15) (SEQ ID NO: 114)

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15) NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS The nucleic acid encoding IL-15/TF/IL-15 was cloned into a modified retrovirus expression vector as described previously (Hughes et al., Hum Gene Ther 16:457-72, 2005). The expression vector encoding IL-15/TF/IL-15 was transfected into CHO-K1 cells. Expression of the expression vector in CHO-K1 cells allowed for secretion of the soluble IL-15/TF/IL-15 single-chain chimeric polypeptide (referred to as 15tl 5), which can be purified by anti-TF antibody affinity and other chromatography methods.

75/75 promotes II -2 R b and common g chain containing 32Όb cell proliferation

IL-15 activity of 15tl 5 was compared with recombinant IL-15 in IL2R{$ and common g chain expressed 32ϋb cells. IL-15 dependent 32ϋb cells were washed five times with IMDM-l0% FBS and seeded to the wells at 2 x 10 ⁴ cells/well. Serial dilutions of 15tl 5 or IL-15 were added to the cells (Figure 170). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by adding 10 mΐ of WST1 to each well in the day 3 and incubating for an additional 3 hours in a CO2 incubator at 37°C. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in Figure 170, 15tl 5 promoted 32ϋb cell proliferation less efficiently as compared to IL-15. The EC50 of 15tl 5 and IL-15 was 161.4 pM and 1.6 pM, respectively.

Purification elution chromatograph of 15tl5 from anti-TF antibody affinity column

15tl 5 harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0.1M acetic acid, pH 2.9. A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 kDa molecular weight cutoff. As shown in

Figure 171, the anti-TF antibody affinity column bound to 15tl 5 which contains TF as a fusion domain. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine, pH 2.5. The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Reduced SDS-PAGE of 75/75

To determine the purity and molecular weight of the protein, 15tl 5 protein sample purified with anti-TF antibody affinity column was analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) method under reduced condition. After electrophoresis, the gel was stained with InstantBlue for about 30 min, followed by destaining overnight in purified water.

To verify that the 15tl 5 protein undergoes glycosylation after translation in CHO cells, a deglycosylation experiment was conducted using the Protein Deglycosylation Mix II kit from New England Biolabs and the manufacturer’s instructions. Figures 172A and 172B show the reduced SDS-PAGE analysis of the sample in non-deglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state. The results showed that the 15tl 5 protein is glycosylated when expressed in CHO cells. After deglycosylation, the purified sample ran with expected molecular weights (50 kDa) in reduced SDS gel. Lane M was loaded with 10 pL of SeeBlue Plus2 Prestained Standard.

Example 87: Stimulation of NK cells in vitro by multi-chain chimeric polypeptide constructs alone or in combination with an anti-TF antibody

A set of experiments was performed to assess the changes in surface phenotype of

NK cells after stimulation with 18tl 5-l2s, l8tl5-l2sl6, and 7tl5-2ls + anti-TF antibody. In these experiments, fresh human leukocytes were obtained from the blood bank and CD56 ⁺ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >90% and confirmed by staining with CD56- BV421, CD16-BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). The cells were counted and resuspended at 0.2 x l0 ⁶ /mL in a 96-well flat-bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were stimulated with: l8tl5-l2s (100 nM); l8tl5-l2sl6 (100 nM); a mixture of single cytokines rhILl5 (50 ng/mL) (Miltenyi), rhILl8 (50 ng/mL) (Invivogen), and l 2 (10 ng/mL) (Peprotech);

7tl 5-21 s + anti-TF antibody (100 nM-50 nM); 7tl 5-21 s (100 nM); or anti-TF antibody (50nM) at 37 °C and 5% CO2 for 16 hours. The next day, the cells were harvested and surface stained for 30 minutes for CD56, CD16, CD25, CD69, CD27, CD62L, NKp30, and NKp44. After surface staining, the cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (IX PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes, the cells were analyzed by flow cytometry (Celesta-BD Bioscience). Figure 173 shows that overnight incubation of purified NK cells with l8tl5-l2s, l8tl5-l2sl6, and 7tl5-2ls + anti-TF antibody resulted in an increase in the percentage of cells expressing CD25, CD69, NKp44, and NKp30 activation markers and a decrease in the percentage of cells expressing CD62L. All activation marker data is from CD56 ⁺ gated lymphocytes.

A set of experiments was performed to assess changes in the surface phenotype of lymphocyte populations after stimulation with l8tl5-l2s, l8tl5-l2sl6, and 7tl5-2ls. In these experiments, fresh human leukocytes were obtained from the blood bank.

Peripheral blood lymphocytes were isolated with the Ficoll-PAQUE Plus (GE

Healthcare) density gradient media. The cells were counted and resuspended at 0.2 x l0 ⁶ /mL in a 96-well flat-bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were stimulated with: l8tl5-l2s (100 nM); 18tl 5- l2s 16 (100 nM), a mixture of single cytokines rhILl5 (50 ng/mL) (Miltenyi), rhILl8 (50 ng/mL) (Invivogen), and rhTL-12 (10 ng/mL) (Peprotech); 7tl 5-21 s (100 nM) + anti-TF antibody (50 nM); 7tl 5- 2ls (100 nM); or anti-TF antibody (50 nM) at 37 °C and 5% CO2 for 16 hours. The next day, the cells were harvested and surface stained for 30 minutes for CD4 or CD8,

CD62L, and CD69. After surface staining, cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (IX PBS (Hyclone) with 0.5% BSA (EMD

Millipore) and 0.001% sodium azide (Sigma)). After two washes, the cells were analyzed by flow cytometry (Celesta-BD Bioscience). Figure 174 shows that overnight incubation of purified lymphocyte populations (CD4 and CD8 T cells) with l8tl5-l2s, l8tl5-l2sl6, or 7tl 5-21 s + anti-TF antibody resulted in an increase in the percentage of

CD8 and CD4 T cells expressing CD69. Additionally, incubation with 7tl 5-21 s + anti- TF antibody resulted in an increase in the percentage of CD8 and CD4 T cells expressing CD62L (Figure 174). Example 88: Induction of proliferation of NK cells in vitro by 7tl5-21s and anti-TF antibody

A set of experiments was performed to determine the effect of 7tl5-2ls or the combination of 7tl 5-21 s + anti-TF antibody on immune cell proliferation. In these experiments, fresh human leukocytes were obtained from the blood bank and CD56 ⁺ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >90% and confirmed by staining with CD56-BV421, CD16- BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). The cells were counted and resuspended in 2 x l0 ⁶ /mL in a 24 well flat bottom plate in 2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were stimulated with: 7tl 5-21 s (100 nM), 7tl 5-21 s + anti-TF antibody (IgGl Ab, 50 nM) or 7tl 5-21 s (100 nM) + anti-TF antibody IgG4 (50 nM) for 5 days. After every 2 days, the cells were resuspended at 2 x l0 ⁶ /mL with fresh media with the multi-chain chimeric polypeptides and in some instances, the anti-TF antibodies described mentioned above. After 5 days, cells were counted using trypan blue to access the fold-expansion (Data shown for 3 healthy donors). Figure 175 shows 7tl 5-21 s + anti-TF antibody treatment provided a greater fold-increase in NK cell expansion than treatment with 7tl5-2ls alone or 7tl 5-21 s + anti- TF antibody IgG4.

A set of experiments was performed to determine NK proliferation following treatment with the combination of 7tl 5-21 s + anti-TF antibody. In these experiments, fresh human leukocytes were obtained from the blood bank and CD56 ⁺ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >90% and confirmed by staining with CD56-BV421, CD16- BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). The cells were counted and resuspended in 2 x l0 ⁶ /mL in a 24-well flat-bottom plate in 2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were stimulated with: 7tl 5-21 s (100 nM) and anti-TF antibody (50 nM) for up to 15 days. After every 2 days, cells were resuspended at 2 x l0 ⁶ /mL with fresh media containing 100 nM 7tl 5-21 s and 50 nM of anti-TF antibody. As the volume of the cultures increased, the cells were transferred to higher volume flasks. At the indicated time points, the cells were counted using trypan blue to access the fold-expansion (data shown for 1 healthy donor). Figure 176 shows that 7tl5-2ls + anti-TF antibody treatment provided a >200-fold increase in NK cell expansion over 2 weeks in culture.

A set of experiments was performed to determine changes in the cell surface phenotype of NK cells expanded following treatment with 7tl 5-21 s + anti-TF antibody. In these experiments, fresh human leukocytes were obtained from the blood bank and CD56 ⁺ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >90% and confirmed by staining with CD56- BV421, CD16-BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). The cells were counted and resuspended in 2 x l0 ⁶ /mL in a 24-well flat-bottom plate in 2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were stimulated with: 7tl 5-21 s (100 nM) and anti-TF antibody (50 nM) for 15 days. After every 2 days, the cells were resuspended at 2 x l0 ⁶ /mL with fresh media containing 100 nM 7tl 5-21 s and 50 nM of anti-TF antibody. As the volume of the cultures increased, the cells were transferred to higher volume flasks. To assess the change in the phenotype of NK cells, expanded cells were stained with antibodies for cell-surface CD56, CD 16, CD25, CD69, CD62L, and CD57 (BioLegend). After surface staining, the cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (IX PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes, the cells were analyzed by flow cytometry (Celesta-BD Bioscience). The cell surface phenotype of NK cells on Day 0 and Day 15 were compared. Figure 177 shows that incubation of NK cells with 7tl 5-21 s + anti-TF antibody resulted in an increase in the percentage of

CDl6 ⁺ CD56 ⁺ NK cells expressing CD25 and CD69, and a decrease in the percentage of CD16 ⁺ CD56 ⁺ NK cells expressing CD62L and CD57.

Example 89: Induction of proliferation of immune cells in vivo

A set of experiments was performed to determine the effect 7tl 5-21 s + anti-TF antibody-expanded NK cells in NSG mice following treatment with 7tl5-2ls, TGFRtl5- TGFRs, and 2t2. In these experiments, fresh human leukocytes were obtained from the blood bank and CD56 ⁺ NK cells were isolated with the Rosette Sep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >90% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). The cells were counted and resuspended in 2 x l0 ⁶ /mL in a 24-well flat- bottom plate in 2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were stimulated with: 7tl 5-21 s (100 nM) and anti-TF antibody (50 nM) for 15 days. After every 2 days, the cells were resuspended at 2 x l0 ⁶ /mL with fresh media containing 100 nM 7tl5-2ls and 50 nM of anti-TF antibody. As the volume of the cultures increased, the cells were transferred to higher volume flasks. The cells were counted using trypan blue to access the fold-expansion. 7tl 5-21 s + anti-TF antibody-expanded NK cells were washed three times in warm HBSS Buffer (Hyclone) at 1000 RPM for 10 minutes at room temperature. The 7tl5-2ls + anti-TF antibody-expanded-NK cells were

resuspended in 10 x l0 ⁶ /0.2 mL HBSS buffer and injected intravenously into the tail vein of NSG mice (NOD scid gamma mouse) (Jackson Laboratories). The transferred NK cells were supported every 48 hours with either 7tl 5-21 s (10 ng/dose, i.p.), TGFRU5- TGFRs (10 ng/dose, i.p.) or 2t2 (10 ng/dose, i.p.) for up to 21 days. Engraftment and persistence of the human 7tl 5-21 s + anti-TF antibody-expanded NK cells were measured every week in blood staining for hCD45, mCD45, hCD56, hCD3, and hCDl6 antibodies by flow cytometry (Celesta-BD Bioscience) (Data represent 3 mice per group). Figure 178 indicates that treatment of mice bearing adoptively-transferred 7tl5-2ls + anti-TF antibody-expanded NK cells with 7tl 5-21 s-, TGFRtl5-TGFRs-, or 2t2-induced expansion and persistence of the adoptively transferred NK cells compared to control treated mice.

Example 90: NK-mediated cytotoxicity following treatment with multi-chain construct + anti-TF antibody

A set of experiments was performed to determine the effective killing of chemical- induced senescent human fibroblasts by 7tl5-2ls + anti-TF antibody-expanded NK cells. In these experiments, human lung fibroblasts (IMR-90 and WI-38) were treated with doxorubicin (0.8mM) for 48 hours. The cells were rested for 48 hours and then treated with doxorubicin (0.8mM) again for 48 hours. Doxorubicin-containing culture medium was changed and the cells were rested in culture medium for 7 days. Figure 179A shows beta galactosidase staining confirming senescence induction. NK cells isolated from healthy donors were expanded as follows: NK cells were treated with 7tl 5-21 s (100 nM) and anti-TF antibody (50 nM) for 15 days. After every 2 days, cells were resuspended at 2 x l0 ⁶ /mL with fresh media containing 100 nM of 7tl 5-21 s and 50 nM of anti-TF antibody. As the volume of the cultures increased, the cells were transferred to higher volume flasks. The cells were counted using trypan blue to access the fold-expansion. On the day of experiment, the target cells were trypsinized, labeled with Cell Trace Violet (CTV), and added to expanded NK cells at an Effector (NK):Target ratio (ET) of 4: 1. The cell mixture was incubated for 20 hours. Non-senescent target fibroblast cells and untreated senescent human fibroblasts were included as controls. After incubation, the cells were trypsinized, washed, and resuspended in Propidium Iodide (PI) solution. The cytotoxicity of the 7tl 5-21 s + anti-TF antibody-expanded NK cells against the chemical-induced senescent human fibroblasts was accessed by flow cytometry based on PI staining of the CTV-labeled cells. Figure 179B shows percent of dead (PI ⁺ ) target cells for non-senescent cells (left two bars) and for senescent cells (right two bars). The results demonstrate that 7tl 5-21 s expanded human NK cells effectively kill non- senescent and senescent cells (Figure 179B).

A set of experiments was performed to assess the effective killing of UV-induced senescent human fibroblasts by 7tl 5-21 s + anti-TF antibody-expanded NK cells. In these experiments, human foreskin fibroblasts (HFF) were grown in 48-well plates (0.1 x 10 ⁶ cells/well) overnight and treated with ultraviolet (UV) B light (3250J/m ² ) twice a day for 5 days. The culture medium was changed every 3 days. After UV exposure, the cells were kept for 2 days, trypsinized, and transferred to T175 flask. After 4 days, the cells were trypsinized and beta galactosidase staining and gene expression (senescence markers and NK ligand) analysis was performed to confirm induction of senescence. Figure 180A shows beta galactosidase staining confirming the induction of senescence. Figure 180B shows gene expression for senescence markers and an NK ligand, which further confirm the induction of senescence.

To assess killing of UV-induced senescent human fibroblasts, NK cells isolated from healthy donors were expanded as follows: NK cells were treated with 7tl5-2ls (100 nM) and anti-TF antibody (50 nM) for 15 days. After every 2 days, the cells were resuspended at 2 x l0 ⁶ /mL with fresh media containing 100 nM of 7tl 5-21 s and 50 nM of anti-TF antibody. As the volume of the cultures increased, the cells were transferred to higher volume flasks. The cells were counted using trypan blue to access the fold- expansion. On the day of experiment, target cells (UV-induced senescent human fibroblasts) were trypsinized, labeled with Cell Trace Violet (CTV), and added to expanded NK cells at an Effector (NK):Target (HFF) ratio of 4: 1. The cell mixture was incubated for 10 hours. Untreated target cells were included as a control. After incubation, the cells were trypsinized, washed, and resuspended in propidium iodide (PI) solution and analyzed by flow cytometry. Figure 180C shows the percentage of dead (PI ⁺ ) target cells for non-senescent cells (two left bars) and for senescent cells (two right bars). The results demonstrate that 7tl5-2ls-expanded human NK cells effectively kill non-senescent and senescent cells. Example 91: Activation and proliferation of NK cells in vitro

Fresh human leukocytes were obtained from the blood bank and CD56 ⁺ NK cells were isolated with the Rosette Sep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >90% and confirmed by staining with CD56-BV421, CD16- BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). The cells were counted and resuspended at 2 x l0 ⁶ /mL in 24-well flat-bottom plates in 2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life

Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone).

The cells were stimulated with the following molecules: 7tl 5-21 s (100 nM), 7tl 5-

2ls (100 nM) + anti-TF antibody (IgGl Ab, 50 nM), or 7tl5-2ls (100 nM) + anti-TF antibody (IgG4 Ab, 50 nM) for 5 days. After every 2 days, the cells were resuspended at 2 x l0 ⁶ /mL with fresh media with the supplemented molecules. After 5 days, the cells were counted using trypan blue to access the fold-expansion (data shown for 3 healthy donors). The results showed that the 7tl 5-21 s + anti-TF antibody (IgGl) treatment provided a greater fold increase in NK cell expansion than treatment with 7tl 5-21 s alone or with 7tl5-2ls + anti-TF antibody (IgG4) (Figure 181).

Fresh human leukocytes were obtained from the blood bank and CD56 ⁺ NK cells were isolated with the Rosette Sep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >90% and confirmed by staining with CD56-BV421, CD16- BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). The cells were counted and resuspended at 2 x l0 ⁶ /mL in 24-well flat-bottom plate in 2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life

Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone). The cells were stimulated with either 7tl5-2ls (100 nM) and anti-TF antibody (IgGl) (50 nM), or with 7tl5-2lsl37L short version (4lBBs) (100 nM) and anti-TF antibody (IgGl) (50 nM) for up to 13 days. After every 2 days, the cells were resuspended at 2 x l0 ⁶ /mL with fresh media with supplemented molecules. As the volume of the cultures increased, the cells were transferred to higher volume flasks. At the indicated time points as shown in Figure 182, the cells were counted using trypan blue to assess the fold-expansion (data shown for 3 healthy donors). The results showed that the 7tl5-2ls + anti-TF antibody (IgGl) treatment provided a >30-fold increase in NK cell expansion over 13 days in culture (Figure 182).

Example 92: Treatment of a subject having a cancer

Daudi tumor cells (Human Burkitt's lymphoma) (ATCC) were cultured in complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). Daudi cells were washed three times in warm HBSS Buffer (Hyclone) at 1000 RPM for 10 minutes at room temperature. The cells were resuspended in 10 x l0 ⁶ /0.2 mL HBSS buffer and injected intravenously into the tail vein of NSG mice (NOD scid gamma mouse) (The Jackson Laboratory). After day 10, bone marrow was harvested from untreated NSG mice to evaluate tumor cell

engraftment. Engraftment of Daudi cells in bone marrow was verified by flow cytometry (Celesta-BD Bioscience) using PE-conjugated anti-human HLA-DR antibody

(BioLegend).

Fresh human leukocytes were obtained from the blood bank and CD56 ⁺ NK cells were isolated with the Rosette Sep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >90% and confirmed by staining with CD56-BV421, CD16- BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). The cells were counted and resuspended at 2 x l0 ⁶ /mL in 24-well flat-bottom plates in 2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life

Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were stimulated with 7tl5-2ls (100 nM) and anti-TF antibody (IgGl) (50 nM) for 15 days. After every 2 days, the cells were resuspended at 2 x l0 ⁶ /mL with fresh media with supplemented molecules. As the volume of the cultures increased, the cells were transferred to higher volume flasks. The cells were counted using trypan blue to assess the fold-expansion.

The 7tl 5-21 s + anti-TF antibody (IgGl)-expanded NK cells were washed three times in warm HBSS Buffer (Hyclone) at 1000 RPM for 10 minutes at room temperature. The NK cells were resuspended in 10 x l0 ⁶ /0.2 mL HBSS buffer and injected

intravenously into the tail vein of NSG mice bearing Daudi tumors on day 10. The transferred NK cells were supported every 48 hours with 2t2 (10 ng/dose, i.p.) for up to 7 days. After day 7 post-NK cell transfer, the percentage of HLA-DR+ Daudi cells in the bone marrow was determined by flow cytometry. The data show that adoptive transfer of the 7tl5-2ls + anti-TF antibody (IgGl)-expanded NK cells resulted in a decrease in Daudi tumor cells in the bone marrow of NSG mice (Figure 183).

Example 93: Improvement of the texture and/or appearance and/or hair

The ApoE ^/_ mice were fed with chow diet (shown as“CD” in Figure 184A) or Western diet (shown as“WD” in Figures 184B-E) from Day 0. On Day 44 (Week 7), the mice were administrated subcutaneously with PBS (Figures 184A and 184B), 3 mg/kg of TGFRtl5-TGFRs (Figure 184C), 2t2 (Figure 195D), or anti-TF antibody (IgG4) (Figure 184E). The pictures were taken one week after treatment on Day 51 (Week 8).

Compared to the PBS control group (Figure 184B), the skin texture was significantly improved in mice fed with a Western diet and treated with 2t2 or TGFRtl5-TGFRs (Figures 184C and 184D). Example 94: Tissue factor coagulation assays following treatment with single-chain or multi-chain chimeric polypeptides

A set of experiments was performed to assess blood coagulation following treatment with single-chain or multi-chain chimeric polypeptides. To initiate the blood coagulation cascade pathway, tissue factor (TF) binds to Factor Vila (FVIIa) to form a TF/FVIIa complex. The TF/FVIIa complex then binds Factor X (FX) and converts FX to FXa.

Factor Vila (FVIIa) activity Assay

One assay to measure blood coagulation involves measuring Factor Vila (FVIIa) activity. This type of assay requires the presence of tissue factor and calcium. The

TF/FVIIa complex activity can be measured by a small substrate or by a natural protein substrate, for example, Factor X (FX). When FX is used as a substrate, phospholipids are also required for TF/FVIIa activity. In this assay, FVIIa activity is determined with FVIIa-specific chromogenic substrate S-2288 (Diapharma, West Chester, OH). The color change of the S-2288 substrate can be measured spectrophotometrically and is proportional to the proteolytic activity of FVIIa (e.g., the TF/FVIIa complex).

In these experiments, the FVIIa activity of the following groups were compared: the 219-amino acid extracellular domain of tissue factor domain (TF219), a multi-chain chimeric polypeptide with a wild-type tissue factor domain, and a multi-chain chimeric polypeptide with a mutant tissue factor domain. The chimeric polypeptides containing mutant tissue factor molecules were constructed with mutations to the TF domain at amino acid sites: Lys20, Ile22, Asp58, Argl35, and Phel40.

In order to assess activity of FVIIa, FVIIa, and TF219 or a TF219 -containing multi - chain chimeric polypeptide were mixed at an equal molar concentration (10 nM) in all wells of a 96-well ELISA plate in a total volume of 70 pL. After incubation for 10 minutes at 37 °C, 10 pL of 8 mM S-2288 substrate was added to start the reaction. The incubation was then kept at 37 °C for 20 minutes. Finally, color change was monitored by reading absorbance at 405 nm. The OD values of different TF/VIIa complexes are shown in Table 1 and Table 2. Table 1 shows a comparison of TF219, 2ltl5-2ls wild-type (WT) and 2ltl 5-21 s mutant (Mut). Table 2 shows a comparison of TF2i9, 2ltl5-TGFRs wild-type (WT), and 2ltl5-TGFRs mutant (Mut). These data show that TF2i9-containing multi-chain chimeric polypeptides (e.g., 2ltl5-2ls-WT, 2ltl5-2ls-Mut, 2ltl5-TGFRS- WT, and 2ltl5-TGFRS-Mut) have lower FVIIa activity than TF219 when the

chromogenic S-2288 was used as a substrate. Notably, the multi-chain chimeric polypeptides containing TF219 mutations showed much lower FVIIa activity when compared to multi-chain chimeric polypeptides containing wild type TF219.

Table 1 FVIIa activity

WT : wild type of TF219, Mut: TF219 containing mutations. Table 2. FVIIa activity

WT: wild type of TF _2i 9,Mut: TF219 containing mutations.

Factor X (FX) Activation Assay

An additional assay to measure blood coagulation involves measuring activation of Factor X (FX). Briefly, TF/VIIa activates blood coagulation Factor X (FX) to Factor Xa (FXa) in the presence of calcium and phospholipids. TF243, which contains the transmembrane domain of TF, has much higher activity in activating FX to FXa than TF219, which does not contain the transmembrane domain. TF/VIIa dependent activation of FX is determined by measuring FXa activity using an FXa-specific chromogenic substrate S-2765 (Diapharma, West Chester, OH). The color change of S-2765 can be monitored spectrophotometrically and is proportional to the proteolytic activity of FXa.

In these experiments, FX activation with a multi-chain chimeric polypeptide (18tl 5-l2s, mouse (m)2ltl5, 2ltl5-TGFRs, and 2ltl5-7s) was compared with a positive control (Innovin) or TF219. TF219 (or TF2i9-containing multi-chain chimeric

polypeptides)/FVIIa complexes were mixed at an equal molar concentration (0.1 nM each) in a volume of 50 pL in round bottom wells of a 96-well ELISA plate, after which 10 pL of 180 nM FX was added. After 15 minutes of incubation at 37 °C, during which time FX was converted to FXa, 8 pL of 0.5 M EDTA (which chelates calcium and thus terminates FX activation by TF/VIIa) was added to each well to stop FX activation.

Next, 10 pL of 3.2 mM S-2765 substrate was added to the reaction mixture.

Immediately, the plate absorbance was measured at 405 nm and was recorded as the absorbance at time 0. The plate was then incubated for 10-20 minutes at 37 °C. The color change was monitored by reading absorbance at 405 nm following the incubation. Results of FX activation as measured by FXa activity using chromogenic substrate S- 2765 are shown in Figure 185. In this experiment, Innovin, which is a commercial prothrombin reagent containing lipidated recombinant human TF243, was used as a positive control for FX activation. Innovin was reconstituted with purified water to about 10 nM of TF243. Next, 0.1 nM TF/VIIa complex was made by mixing an equal volume of 0.2nM of FVIIa with 0.2 nM of Innovin. Innovin demonstrated very potent FX activation activity, while TF219 and TF2i9-containing multi-chain chimeric polypeptides had very low FX activation activity, confirming that TF219 is not active in a TF/FVIIa complex for activating natural substrate FX in vivo.

Prothrombin Time Test

A third assay to measure blood coagulation is the prothrombin time (PT) test, which measures blood clotting activity. Here, the PT test was performed using commercially available normal human plasma (Ci-Trol Coagulation Control, Level I).

For a standard PT test, clot reactions were initiated by addition of Innovin, a lipidated recombinant human TF243, in the presence of calcium. Clotting time was monitored and reported by STart PT analyzer (Diagnostica Stago, Parsippany, N.J.). PT assays were started by injecting 0.2 mL of various dilutions of Innovin diluted in PT assay buffer (50 mM Tris-HCl, pH 7.5, 14.6 mM CaCb, 0.1% BSA) into cuvettes containing 0.1 mL of normal human plasma prewarmed at 37 °C. In the PT assay, shorter PT time (clotting time) indicates a higher TF-dependent clotting activity while longer PT (clotting time) means lower TF-dependent clotting activity.

As seen in Figure 186, addition of different amounts of Innovin (e.g., Innovin reconstituted with purified water equivalent to 10 nM of lipidated recombinant human TF243 was considered to be 100% Innovin) to the PT assay demonstrated a dose-response relationship, where lower concentrations of TF243 resulted in a longer PT time (lower clotting activity). For example, 0.001% Innovin had a PT time greater than 110 seconds, which was almost the same as buffer alone.

In another experiment, the PT test was conducted on TF219 and multi-chain chimeric polypeptides including: 18tl 5-l2s, 7tl5-2ls, 2ltl5-TGFRs-WT, and 21U5- TGFRs-Mut. Figure 187 show that TF219 and TF2i9-containing multi-chain chimeric polypeptides (at a concentration of 100 nM) had prolonged PT times indicating extremely low or no clotting activity. Studies were also conducted to evaluate whether incubating the multi-chain chimeric polypeptides in the presence of other cells carrying receptors for the cytokine components of the multi-chain chimeric polypeptide (32ϋb or human PBMCs) would affect the clotting time in the PT assay. To examine whether cells that express IL-15 receptor (32ϋb cells) or IL-15 and IL-21 receptors (PBMCs) would bind IL-15 - containing multi-chain chimeric polypeptides to mimic natural TF as a cellular FVIIa receptor, TF2i9-containing multi-chain chimeric polypeptides (at a concentration of 100 nM for each molecule) were diluted in the PT assay buffer and preincubated with 32ϋb cells (at 2 x 10 ⁵ cells/mL) or PBMC (at 1 x 10 ⁵ cells/mL) for 20-30 minutes at room temperature. The PT assay was then conducted as described above. Figures 188 and 189 shows that TF219 and TF2i9-containing multi-chain chimeric polypeptides mixed with 32ϋb cells (Figure 188) or PBMC (Figure 189) at a final concentration of 100 nM had prolonged PT times similar to 0.001-0.01% Innovin (equivalent to 0.1 pM to 1.0 pM of TF243). Expressed in percentage of relative TF243 activity, TF2i9-containing multi-chain chimeric polypeptides had 100,000 to 1,000,000 times lower TF dependent clotting activity when compared to Innovin. This demonstrated that TF2i9-containing multi-chain chimeric polypeptides had extremely low or no TF-dependent clotting activity, even while the molecules were bound to an intact cell membrane surface, such as 32ϋb or PBMCs. Example 95: Characterization of 7tl5-21sl37L (long version)

The nucleic acid sequence of the 7tl 5 construct (including signal peptide sequence) is as follows (SEQ ID NO: 144):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC

(Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AG AT C AAGACCTGCTGGAAC AAGATCCTGATGGGC ACC AAGGAGC AT

(Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT T G AGT GC AT GGGC C A AG A A A AGGGC G AGT TC C GGG AG

(Human IL-15)

AACTGGGT GAACGT C ATC AGCGATTT AAAGAAGATCGAAGATTT AATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGT GCGAAGAGCTGGAGGAGAAGAAC AT C AAGGAGTTTCTGC AATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7tl 5 fusion protein (including the leader sequence) is as follows (SEQ ID NO: 143): {Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRA ARKLRQFLKMN S T GDFDLHLLK V SEGTTILLNCTGQ VKGRKP A AL GE AQPTK SLEENK SLKEQKKLNDLCFLKRLLQEIKT C WNKILMGTKEH

(Human Tissue Factor 219)

S GTTNT V A A YNLT WK S TNFKTILEWEPKP VN Q V YT V QIS TK SGD WK SKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQ SFEQ V GTKVNVT VEDERTL VRRNNTFL SLRD VF GKDLI YTL YYW K S S S S GKKT AKTNTNEFLID VDKGEN Y CF S V Q A VIP SRT VNRK S TD SP VECMGQE KGEFRE

(Human IL-15)

NW VN VI SDLKKIEDLIQ SMHID ATL YTE SD VHP S CK VT AMKCFLLELQ VI S LESGD ASIHDTVENLIIL ANN SLS SNGNVTESGCKECEELEEKNIKEFLQSF VHIV Q MFINTS

The nucleic acid sequence of the 2l sl37L construct (including signal peptide sequence) is as follows (SEQ ID NO: 172):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC

(Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCC AAGGAGTTCCTGGAGAGGTTC AAGTCCCTGCTGC AGAAGAT GAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

((G4S)3 linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

(Human CD137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGAC CTGCGGC AGGGC AT GTTT GCGC AGCTGGT GGCCC AAAAT GTTCTGCTGATCG ATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGAC GGGGGGC C T G AGC T AC A A AG AGG AC AC G A AGG AGC T GGT GGT GGC C A AGGC TGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCG AGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCT GCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGA GGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCG GCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTG GCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAA ATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence of 2l sl37L fusion protein (including the leader sequence) is as follows (SEQ ID NO: 171):

{Signal peptide)

MKWVTFISLLFLF S SAYS

(Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEW S AF SCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHL S SRTHGSED S

(Human IL-15R a sushi domain)

ITCPPPMS VEHADIWVKS Y SLY SRERYICN SGFKRK AGT S SLTEC VLNKAT NVAHWTTPSLKCIR ((G4S)3 linker )

GGGGSGGGGSGGGGS

(Human CD137L)

REGPEL SPDDP AGLLDLRQGMF AQL VAQNVLLIDGPL S W Y SDPGL AGV S LTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSA AGAAAL ALTVDLPP AS SEARN S AF GF QGRLLHLS AGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSPRSE

The following experiment was conducted to evaluate whether the CD137L portion in 7tl5-2lsl37L was intact to bind to CD137 (4.1BB). On day 1, a 96-well plate was coated with 100 pL (2.5 pg/mL) of GAH IgG Fc (G-102-C, R&D Systems) in R5 (coating buffer), overnight. On day 2, the plates were washed three times and blocked with 300 pL of 1% BSA in PBS at 37°C for 2 hrs. 10 ng/ml of 4. lBB/Fc (838-4B, R&D Systems) was added at 100 pl/well for 2 hrs at room temperature. Following three washes, 7tl5-2lsl37L (long version) or 7tl5-2lsl37Ls (short version) was added starting at 10 nM, or recombinant human 4.1BBL starting at l80ng/mL, with 1/3 dilution, followed by incubation at 4°C overnight. On day 3, the plates were washed three times, and 500 ng/mL of biotinylate-goat anti-human 4.1BBL (BAF2295, R&D Systems) was applied at 100 pL per well, followed by incubation at RT for 2 hrs. The plates were washed three times, and incubated with 0.25 pg/mL of HRP-SA (Jackson

ImmuneResearch) at 100 pL per well for 30 min. The plates were then washed three times, and incubated with 100 pL of ABTS for 2 mins at RT. The results were read at 405 nm. As shown in Figure 190, both 7tl5-2lsl37L (long version) and 7tl5-2lsl37L (short version) could interact with 4. lBB/Fc (dark diamond and gray square) compared to the recombinant human 4.1BB ligand (rhCDl37L, light gray star). 7tl5-2lsl37L (long version) (dark diamond) interacted better with 4. lBB/Fc as compared to 7tl5-2lsl37L (short version) (gray square).

The following experiments were conducted to evaluate whether the components IL7, IL21, IL15, and 4.1BBL in 7tl5-2lsl37L (long version) were intact to be detected by the individual antibody using ELISA. A 96-well plate was coated with 100 pL (4 pg/mL) of anti-TF (human IgGl) in R5 (coating buffer) and incubated at RT for 2 hrs. The plates were washed three times, and blocked with 100 pL of 1% BS A in PBS.

Purified 7tl5-2lsl37L (long version) was added starting at 10 nM, and at 1/3 dilution, followed by incubation at RT for 60 min. The plates were washed three times, and 500 ng/mL of biotinylate-anti-IL7 (506602, R&D Systems), 500 ng/mL of biotinylate-anti- IL21 (13-7218-81, R&D Systems), 50 ng/mL of biotinylate-anti-ILl5 (BAM247, R&D Systems), or 500 ng/ml of biotinylate-goat anti-human 4.1BBL (BAF2295, R&D

Systems) was added per well and incubated at room temperature for 60 min. The plates were washed three times and incubated with 0.25 pg/mL of HRP-SA (Jackson

ImmunoResearch) at 100 pL per well for 30 min at RT. The plates were washed four times, and incubated with 100 pL of ABTS for 2 mins at room temperature. The absorbance results were read at 405 nm. As shown in Figure 191 A-191D, the

components including IL7, IL21, IL15, and 4.1BBL in 7tl5-2lsl37L (long version) were detected by the individual antibodies.

The following experiment was conducted to evaluate the activity of IL 15 in 7tl 5-

2lsl37L (long version) and 7tl5-2lsl37L (short version). The ability of 7tl5-2lsl37L (long version) and 7tl5-2lsl37L (short version) to promote proliferation of IL2Ra y- expressing CTLL2 cells was compared with that of recombinant IL15. IL15 dependent CTLL2 cells were washed five times with IMDM-l0% FBS and seeded to the wells at 2 x 10 ⁴ cells/well. Serially diluted 7tl5-2lsl37L (long version), 7tl5-2lsl37L (short version), or IL15 were added to the cells. Cells were incubated in a CO2 incubator at 37 °C for 3 days. Cell proliferation was detected by adding 20 pL of PrestoBlue (A13261, ThermoFisher) to each well on day 3 and incubated for an additional 4 hours in a CO2 incubator at 37 °C. Raw absorbance at 570-610 nm was read in a micro-titer plate reader. As shown in Figure 192, 7tl5-2lsl37L (long version), 7tl5-2lsl37L (short version), and IL15 all promoted CTLL2 cell proliferation. The EC50 of 7tl5-2lsl37L (long version), 7tl5-2lsl37L (short version), and IL15 is 51.19 pM, 55.75 pM, and 4.947 pM, respectively. Example 96: Induction of Treg cells by 2t2

The peripheral blood mononuclear cells (PBMC) of a heathy donor (Donor 163) were isolated from 5 mL of whole blood huffy coats by Ficoll Paque Plus (GE17144003). The PBMC were then lysed with ACK to remove red blood cells. Cells were washed with IMDM-lO% FBS and counted. 1.8 xlO ⁶ cells (100 pL/tube) were seeded to the flow tubes and incubated with 50 pL of descending 2t2 or IL2 (15000, 1500, 150, 15, 1.5,

0.15, or 0 pM) and 50 pLof pre-staining antibodies (anti-CD8-BV605 and anti-CDl27- AF647). Cells were incubated for 30 min at 37 °C in water bath. 200 pL of pre-warmed BD Phosflow Fix Buffer I (Cat# 557870, Becton Dickinson Biosciences) was added for 10 min at 37° C in water bath to stop the stimulation. Cells (4.5 xlO ⁵ cells/lOO pL) were transferred to a V-shape 96-well plate and were spun down followed by permeabilization with 100 pL of -20 °C pre-cooled BD Phosflow Perm Buffer III (Cat# BD Biosciences) for 30 min on ice. The cells were then extensively washed x2 with 200 pL of FACS buffer and stained with a panel of fluorescent antibodies (anti-CD25-PE, CD4-PerCP- Cy5.5, CD56-BV421, CD45RA-PE-Cy7 and pSTAT5a-AF488) to distinguish between different lymphocyte subpopulations and evaluate the pSTAT5a status. Cells were spun down and resuspended in 200 pL of FACS buffer for FACSCelesta analysis. As sown in Figure Cl A, 6 pM of 2t2 was sufficient to induce the phosphorylation of Stat5a in CD4 ⁺ CD25 ^M Treg cells while 43.11 pM of IL-2 was required to induce phosphorylation of Stat5a in the same population of lymphocytes. In contrast, 2t2 was less active (Figure

193B) or equally active (Figure 193C) as compared to IL2 in inducing phosphorylation of Stat5a in CD4 ⁺ CD25 con and CD8 Tcon cells. These results suggest that 2t2 is superior as compared to IL2 in activating Treg in human PBMC, and that 2t2 demonstrates increased Treg selectivity compared to IL-2 in human blood lymphocyte pStat5a responses.

Example 97: Differentiation of the Immune Cell into a Memory-Like Immune Cell

Fresh human leukocytes were obtained from the blood bank and CD56 ⁺ NK cells were isolated with the Rosette Sep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16- BV510, CD25-PE, CD69-APCFire750 (BioLegend). The cells were counted and resuspended at a density of 2 x 10 ⁶ cells/mL in RPMI 1640 medium (Gibco)

supplemented with 2 mM L-glutamine (Thermo Life Technologies), antibiotics

(penicillin, 10,000 units/mL; streptomycin, 10,000 pg/mL; Thermo Life Technologies), and 10% FBS (Hyclone). The cells (1 mL) were transferred into a 24-well flat bottom plate, and subjected to either: no treatment, or expanded with 7tl5-2ls + anti -tissue factor (TF)-antibody (IgGl) (50 nM) for 14 days with medium. The cells were replenished with fresh 7tl 5-21 s + anti-TF-antibody (IgGl) (50 nM) to keep the cell density at

approximately 1 x 10 ⁶ cells/mL.

Unexpanded NK cells to treatment groups were used as positive controls for full DNA methylation levels (Data not shown). NK cells were pelleted (1 x 10 ⁶ ), and genomic DNA (nDNA) isolated using the QIAamp UCP DNA Micro Kit (Qiagen). 500 ng of purified nDNA was subjected to sodium bisulfite treatment using the EZ DNA Methylation-Direct kit (Zymo Research) according to the manufacturer’s protocol.

Bisulfite treatment introduces methylation-dependent changes in the DNA with demethylated cytosines being converted into uracil, whereas methylated cytosines remain unchanged. The bi sulfite-treated nDNA (10-50 ng) was used as template to PCR amplify a 228 bp region of the IFNy promoter containing two CpG sites (CpG -186 and CpG -54, position relative to the transcription start site, TSS), known to be heavily regulated by DNA methylation in T cells, using the Pyromark PCR kit (Qiagen) with the forward primer IFNG127F (5 '-AT GGT AT AGGT GGGT AT AAT GG-3 ') (SEQ ID NO: 30) and the biotinylated reverse primer IFNG355R-bio (biotin-5'-

CAATATACTACACCTCCTCTAACTAC-3 ') (SEQ ID NO: 43) (GENEWIZ). The PCR conditions were 15 min at 95 °C, 48 cycles of 30 sec at 95 °C, 30 sec at 56 °C, 60 sec at 72 °C followed by 10 min at 72 °C. The integrity and quality of the PCR amplified products were visualized on a 1.2% TAE agarose gel. The DNA methylation status of these two CpG sites was determined by pyrosequencing, which is the gold standard technique to quantitatively measure DNA methylation at single CpG-site.

Pyrosequencing reactions were performed at Johns Hopkins University Genetic

Resources Core Facility using the DNA sequencing primers C186-IFNG135F (5'- GGTGGGTATAATGGG-3 ') (SEQ ID NO: 14) and C54-IFNG261F (5'-

ATTATTTTATTTTAAAAAATTTGTG-3 ') (SEQ ID NO: 15), specific to the CpG sites -186 and -54, respectively. Commercially available non-methylated and methylated DNA (Zymo Research) were used as controls for DNA methylation. The methylation percentages of the two CpG sites (-186 and -54) were pooled for each treatment. The percent difference in DNA methylation was calculated relative to the levels of DNA methylation at the two CpG sites observed in unexposed NK cells.

Analysis of the DNA methylation status of these two IFNy CpG sites revealed higher levels of DNA demethylation in NK cells supported by 7tl 5-21 s + anti-TF- antibody compared to unexposed NK cells (Figure 194). These 7tl 5-21 s + anti-TF- antibody supported NK cells exhibited 47.70% ± 11.76 difference in DNA methylation (i.e., demethylation) compared to unexposed NK cells. The DNA methylation levels of these two IFNY CpG sites correlated with increased expression of IFNy following treatment with 7tl5-2ls + anti-TF-antibody. These data suggest that long-term exposure of NK cells (14 days expansion in culture) with a combination regimen of 7tl 5-21 s + anti-TF-antibody is able to induce DNA demethylation of the two hypomethylated IFNy CpG sites (-186 and -54) and that 7tl 5-21 s + anti-TF-antibody (IgGl) can epigenetically reprogram gene expression of IFNy via DNA demethylation of CpG sites leading to interconversion of NK cells into innate immune memory NK cells.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Exemplary Embodiments

Embodiment Al. A single-chain chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a second target-binding domain.

Embodiment A2. The single-chain chimeric polypeptide of embodiment Al, wherein the first target-binding domain and the soluble tissue factor domain directly abut each other.

Embodiment A3. The single-chain chimeric polypeptide of embodiment Al, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain. Embodiment A4. The single-chain chimeric polypeptide of any one of embodiments A1-A3, wherein the soluble tissue factor domain and the second target- binding domain directly abut each other.

Embodiment A5. The single-chain chimeric polypeptide of any one of embodiments A1-A3, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the second target-binding domain.

Embodiment A6. The single-chain chimeric polypeptide of embodiment Al, wherein the first target-binding domain and the second target-binding domain directly abut each other.

Embodiment A7. The single-chain chimeric polypeptide of embodiment Al, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the second target-binding domain. Embodiment A8. The single-chain chimeric polypeptide of embodiment A6 or A7, wherein the second target-binding domain and the soluble tissue factor domain directly abut each other. Embodiment A9. The single-chain chimeric polypeptide of embodiment A6 or

A7, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the second target-binding domain and the soluble tissue factor domain.

Embodiment A10. The single-chain chimeric polypeptide of any one of embodiments A1-A9, wherein the first target-binding domain and the second target- binding domain bind specifically to the same antigen.

Embodiment Al l. The single-chain chimeric polypeptide of embodiment A10, wherein the first target-binding domain and the second target-binding domain bind specifically to the same epitope.

Embodiment A12. The single-chain chimeric polypeptide of embodiment Al l, wherein the first target-binding domain and the second target-binding domain comprise the same amino acid sequence.

Embodiment A13. The single-chain chimeric polypeptide of any one of embodiments A1-A9, wherein the first target-binding domain and the second target- binding domain bind specifically to different antigens. Embodiment A14. The single-chain chimeric polypeptide of any one of embodiments A1-A13, wherein one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain.

Embodiment A15. The single-chain chimeric polypeptide of embodiment A14, wherein the first target-binding domain and the second target-binding domain are each an antigen-binding domain. Embodiment A16. The single-chain chimeric polypeptide of embodiment A13, wherein antigen-binding domain comprises a scFv or a single domain antibody.

Embodiment A17. The single-chain chimeric polypeptide of any one of embodiments A1-A16, wherein one or both of the first target-binding domain and the second target-binding domain bind to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin,

CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-pRII), a ligand of TGF-pRIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-l 5, a receptor for IL- 17, a receptor for IL-l 8, a receptor for IL-21, a receptor for PDGF-D, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.

Embodiment A18. The single-chain chimeric polypeptide of any one of embodiments A1-A16, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein.

Embodiment A19. The single-chain chimeric polypeptide of embodiment A18, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL- 17, IL- 18, IL-21 , PDGF-D, SCF, FLT3L, MICA, MICB, and a ULP 16-binding protein. Embodiment A20. The single-chain chimeric polypeptide of any one of embodiments A1-A16, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, soluble cytokine receptor, or soluble cell surface receptor.

Embodiment A21. The single-chain chimeric polypeptide of embodiment A20, wherein the soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor is a soluble TGF-b receptor II (TGF-PRII), a soluble TGF-PRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28.

Embodiment A22. The single-chain chimeric polypeptide of any one of embodiments A1-A21, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment A23. The single-chain chimeric polypeptide of embodiment A22, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1. Embodiment A24. The single-chain chimeric polypeptide of embodiment A23, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment A25. The single-chain chimeric polypeptide of embodiment A24, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1.

Embodiment A26. The single-chain chimeric polypeptide of any one of embodiments A22-A25, wherein the soluble human tissue factor domain does not comprise one or more of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment A27. The single-chain chimeric polypeptide of embodiment A26, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. Embodiment A28. The single-chain chimeric polypeptide of any one of embodiments A1-A27, wherein the soluble tissue factor domain is not capable of binding Factor Vila. Embodiment A29. The single-chain chimeric polypeptide of any one of embodiments A1-A28, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment A30. The single-chain chimeric polypeptide of any one of embodiments A1-A29, wherein the single-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

Embodiment A31. The single-chain chimeric polypeptide of any one of embodiments A1-A30, wherein the single-chain chimeric polypeptide further comprises one or more additional target-binding domains at its N- and/or C-terminus.

Embodiment A32. The single-chain chimeric polypeptide of embodiment A31, wherein the single-chain chimeric polypeptide comprises one or more additional target- binding domains at its N-terminus.

Embodiment A33. The single-chain chimeric polypeptide of embodiment A32, wherein one or more additional target-binding domains directly abuts the first target- binding domain, the second target-binding domain, or the soluble tissue factor domain. Embodiment A34. The single-chain chimeric polypeptide of embodiment A33, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target-binding domains and the first target- binding domain, the second target-binding domain, or the soluble tissue factor domain. Embodiment A35. The single-chain chimeric polypeptide of embodiment A31, wherein the single-chain chimeric polypeptide comprises one or more additional target- binding domains at its C-terminus. Embodiment A36. The single-chain chimeric polypeptide of embodiment A35, wherein one of the one or more additional target-binding domains directly abuts the first target-binding domain, the second target-binding domain, or the soluble tissue factor domain. Embodiment A37. The single-chain chimeric polypeptide of embodiment A35, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target-binding domains and the first target- binding domain, the second target-binding domain, or the soluble tissue factor domain. Embodiment A38. The single-chain chimeric polypeptide of embodiment A31, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-terminus and the C-terminus.

Embodiment A39. The single-chain chimeric polypeptide of embodiment A38, wherein one of the one or more additional antigen binding domains at the N-terminus directly abuts the first target-binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment A40. The single-chain chimeric polypeptide of embodiment A38, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional antigen-binding domains at the N-terminus and the first target-binding domain, the second target-binding domain, or the soluble tissue factor domain. Embodiment A41. The single-chain chimeric polypeptide of embodiment A38, wherein one of the one or more additional antigen binding domains at the C-terminus directly abuts the first target-binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment A42. The single-chain chimeric polypeptide of embodiment A38, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional antigen-binding domains at the C-terminus and the first target-binding domain, the second target-binding domain, or the soluble tissue factor domain. Embodiment A43. The single-chain chimeric polypeptide of any one of embodiments A31-A42, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. Embodiment A44. The single-chain chimeric polypeptide of embodiment A43, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope. Embodiment A45. The single-chain chimeric polypeptide of embodiment A44, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence. Embodiment A46. The single-chain chimeric polypeptide of embodiment A43, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same antigen.

Embodiment A47. The single-chain chimeric polypeptide of embodiment A46, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same epitope. Embodiment A48. The single-chain chimeric polypeptide of embodiment A47, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each comprise the same amino acid sequence. Embodiment A49. The single-chain chimeric polypeptide of any one of embodiments A31-A42, wherein the first target-binding domain, the second target- binding domain, and the one or more additional target-binding domains bind specifically to different antigens. Embodiment A50. The single-chain chimeric polypeptide of any one of embodiments A31-A49, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen binding domain. Embodiment A51. The single-chain chimeric polypeptide of embodiment A50, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen-binding domain.

Embodiment A52. The single-chain chimeric polypeptide of embodiment A51, wherein antigen-binding domain comprises a scFv or a single domain antibody.

Embodiment A53. The single-chain chimeric polypeptide of any one of embodiments A31-A52, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD 19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CE AC AM5, a UL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF- RII), a ligand of TGF- RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-D, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD28. Embodiment A54. The single-chain chimeric polypeptide of any one of embodiments A31-A52, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. Embodiment A55. The single-chain chimeric polypeptide of embodiment A54, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-l 7, IL-l 8, IL-21, PDGF-D, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment A56. The single-chain chimeric polypeptide of any one of embodiments A31-A52, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor.

Embodiment A57. The single-chain chimeric polypeptide of embodiment A56, wherein the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28. Embodiment A58. The single-chain chimeric polypeptide of any one of embodiments A1-A57, wherein the single-chain chimeric polypeptide further comprises a signal sequence at its N-terminal end. Embodiment A59. The single-chain chimeric polypeptide of any one of embodiments A1-A58, wherein the single-chain chimeric polypeptide further comprises a peptide tag positioned at the N-terminal end or the C-terminal end of the single-chain chimeric polypeptide. Embodiment A60. A composition comprising any of the single-chain chimeric polypeptides of embodiments A1-A59.

Embodiment A61. The composition of embodiment A60, wherein the

composition is a pharmaceutical composition.

Embodiment A62. A kit comprising at least one dose of the composition of embodiment A60 or A61.

Embodiment A63. Nucleic acid encoding any of the single-chain chimeric polypeptides of any one of embodiments A1-A59.

Embodiment A64. A vector comprising the nucleic acid of embodiment A63.

Embodiment A65. The vector of embodiment A64, wherein the vector is an expression vector.

Embodiment A66. A cell comprising the nucleic acid of embodiment A63 or the vector of embodiment A64 or A65. Embodiment A67. A method of producing a single-chain chimeric polypeptide, the method comprising: culturing the cell of embodiment A66 in a culture medium under conditions sufficient to result in the production of the single-chain chimeric polypeptide; and

recovering the single-chain chimeric polypeptide from the cell and/or the culture medium.

Embodiment A68. A single-chain chimeric polypeptide produced by the method of embodiment A67.

Embodiment A69. The single-chain chimeric polypeptide of embodiment A26, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 5.

Embodiment A70. The single-chain chimeric polypeptide of embodiment A69, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5.

Embodiment A71. The single-chain chimeric polypeptide of embodiment A70, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 5.

Embodiment A72. The single-chain chimeric polypeptide of embodiment A71, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 5. Embodiment A73. The single-chain chimeric polypeptide of embodiment A26, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 6.

Embodiment A74. The single-chain chimeric polypeptide of embodiment A73, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 6. Embodiment A75. The single-chain chimeric polypeptide of embodiment A74, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 6. Embodiment A76. The single-chain chimeric polypeptide of embodiment A75, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 6.

Embodiment Bl. A single-chain chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a second target-binding domain,

wherein:

the first target-binding domain and the second target-binding domain each specifically bind to an IL-2 receptor; or

the first target-binding domain and the second target-binding domain each specifically bind to an IL-15 receptor.

Embodiment B2. The single-chain chimeric polypeptide of embodiment Bl, wherein the first target-binding domain and the soluble tissue factor domain directly abut each other. Embodiment B3. The single-chain chimeric polypeptide of embodiment Bl, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain.

Embodiment B4. The single-chain chimeric polypeptide of any one of embodiments B1-B3, wherein the soluble tissue factor domain and the second target- binding domain directly abut each other.

Embodiment B5. The single-chain chimeric polypeptide of any one of embodiments B1-B3, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the second target-binding domain.

Embodiment B6. The single-chain chimeric polypeptide of embodiment Bl, wherein the first target-binding domain and the second target-binding domain directly abut each other. Embodiment B7. The single-chain chimeric polypeptide of embodiment Bl, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the second target-binding domain. Embodiment B8. The single-chain chimeric polypeptide of embodiment B6 or B7, wherein the second target-binding domain and the soluble tissue factor domain directly abut each other.

Embodiment B9. The single-chain chimeric polypeptide of embodiment B6 or B7, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the second target-binding domain and the soluble tissue factor domain.

Embodiment B 10. The single-chain chimeric polypeptide of any one of embodiments B1-B9, wherein both the first target-binding domain and the second target- binding domain is a soluble interleukin protein.

Embodiment Bl 1. The single-chain chimeric polypeptide of embodiment B 10, wherein the first target-binding domain and the second target-binding domain is a soluble IL-2 protein.

Embodiment B 12. The single-chain chimeric polypeptide of embodiment Bl 1, wherein the soluble IL-2 protein is a soluble human IL-2 protein.

Embodiment B 13. The single-chain chimeric polypeptide of embodiment B 12, wherein the soluble human IL-2 protein comprises SEQ ID NO: 28.

Embodiment B 14. The single-chain chimeric polypeptide of embodiment B 10, wherein the first target-binding domain and the second target-binding domain is a soluble IL-15 protein. Embodiment B 15. The single-chain chimeric polypeptide of embodiment B 14, wherein the soluble IL-15 protein is a soluble human IL-15 protein.

Embodiment B 16. The single-chain chimeric polypeptide of embodiment B 15, wherein the soluble human IL-15 protein comprises SEQ ID NO: 39.

Embodiment B 17. The single-chain chimeric polypeptide of any one of embodiments B1-B16, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment B 18. The single-chain chimeric polypeptide of embodiment B 17, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1. Embodiment B 19. The single-chain chimeric polypeptide of embodiment B 18, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment B20. The single-chain chimeric polypeptide of embodiment B 19, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1.

Embodiment B21. The single-chain chimeric polypeptide of any one of embodiments B17-B20, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment B22. The single-chain chimeric polypeptide of embodiment B21, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment B23. The single-chain chimeric polypeptide of any one of embodiments B1-B22, wherein the soluble tissue factor domain is not capable of binding Factor Vila. Embodiment B24. The single-chain chimeric polypeptide of any one of embodiments B1-B23, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. Embodiment B25. The single-chain chimeric polypeptide of any one of embodiments B1-B24, wherein the single-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

Embodiment B26. The single-chain chimeric polypeptide of any one of embodiments B1-B25, wherein the single-chain chimeric polypeptide further comprises one or more additional target-binding domains at its N- and/or C-terminus.

Embodiment B27. The single-chain chimeric polypeptide of embodiment B26, wherein the single-chain chimeric polypeptide comprises one or more additional target- binding domains at its N-terminus.

Embodiment B28. The single-chain chimeric polypeptide of embodiment B27, wherein one or more additional target-binding domains directly abuts the first target- binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment B29. The single-chain chimeric polypeptide of embodiment B28, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target-binding domains and the first target- binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment B30. The single-chain chimeric polypeptide of embodiment B26, wherein the single-chain chimeric polypeptide comprises one or more additional target- binding domains at its C-terminus. Embodiment B31. The single-chain chimeric polypeptide of embodiment B30, wherein one of the one or more additional target-binding domains directly abuts the first target-binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment B32. The single-chain chimeric polypeptide of embodiment B30, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target-binding domains and the first target- binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment B33. The single-chain chimeric polypeptide of embodiment B26, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-terminus and the C-terminus.

Embodiment B34. The single-chain chimeric polypeptide of embodiment B33, wherein one of the one or more additional antigen binding domains at the N-terminus directly abuts the first target-binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment B35. The single-chain chimeric polypeptide of embodiment B33, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional antigen-binding domains at the N-terminus and the first target-binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment B36. The single-chain chimeric polypeptide of embodiment B33, wherein one of the one or more additional antigen binding domains at the C-terminus directly abuts the first target-binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment B37. The single-chain chimeric polypeptide of embodiment B33, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional antigen-binding domains at the C-terminus and the first target-binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment B38. The single-chain chimeric polypeptide of any one of embodiments B26-B37, wherein each of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to an IL-2 receptor or an IL-15 receptor.

Embodiment B39. The single-chain chimeric polypeptide of embodiment B38, wherein each of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

Embodiment B40. The single-chain chimeric polypeptide of any one of embodiments B26-B37, wherein the one or more additional target-binding domains is an antigen-binding domain.

Embodiment B41. The single-chain chimeric polypeptide of embodiment B40, wherein the antigen-binding domain comprises a scFv or a single domain antibody.

Embodiment B42. The single-chain chimeric polypeptide of any one of embodiments B26-B37, B40, and B41, wherein the one or more additional target-binding domains bind specifically to a target selected from the group consisting of: CDl6a,

CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL- 10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a,

CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a EIL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF- DD, a ligand of TGF-b receptor II (TGF-PRII), a ligand of TGF- RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of

NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-l 2, a receptor for IL-l 5, a receptor for IL-l 7, a receptor for IL-l 8, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.

Embodiment B43. The single-chain chimeric polypeptide of any one of embodiments B6-B37, B40, and B41, wherein the one or more additional target-binding domains is a soluble interleukin or cytokine protein.

Embodiment B44. The single-chain chimeric polypeptide of embodiment B43, wherein the soluble interleukin or cytokine protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

Embodiment B45. The single-chain chimeric polypeptide of any one of embodiments B6-B37, B40, and B41, wherein the one or more additional target-binding domains is a soluble interleukin or cytokine receptor. Embodiment B46. The single-chain chimeric polypeptide of embodiment B45, wherein the soluble receptor is a soluble TGF-b receptor II (TGF-PRII) and a soluble TGF- RIII.

Embodiment B47. The single-chain chimeric polypeptide of any one of embodiments B1-B46, wherein the single-chain chimeric polypeptide further comprises a signal sequence at its N-terminal end.

Embodiment B48. The single-chain chimeric polypeptide of any one of embodiments B1-B47, wherein the single-chain chimeric polypeptide further comprises a peptide tag positioned at the N-terminal end or the C-terminal end of the single-chain chimeric polypeptide. Embodiment B49. A composition comprising any of the single-chain chimeric polypeptides of embodiments B1-B48.

Embodiment B50. The composition of embodiment B49, wherein the composition is a pharmaceutical composition.

Embodiment B51. A kit comprising at least one dose of the composition of embodiment B49 or B50.

Embodiment B52. A nucleic acid encoding any of the single-chain chimeric polypeptides of any one of embodiments B1-B48.

Embodiment B53. A vector comprising the nucleic acid of embodiment B52.

Embodiment B54. The vector of embodiment B53, wherein the vector is an expression vector.

Embodiment B55. A cell comprising the nucleic acid of embodiment B52 or the vector of embodiment B53 or B54.

Embodiment B56. A method of producing a single-chain chimeric polypeptide, the method comprising:

culturing the cell of embodiment B55 in a culture medium under conditions sufficient to result in the production of the single-chain chimeric polypeptide; and

recovering the single-chain chimeric polypeptide from the cell and/or the culture medium.

Embodiment B57. A single-chain chimeric polypeptide produced by the method of embodiment B56. Embodiment B58. The single-chain chimeric polypeptide of embodiment B21, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 5. Embodiment B59. The single-chain chimeric polypeptide of embodiment B58, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5.

Embodiment B60. The single-chain chimeric polypeptide of embodiment B59, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 5.

Embodiment B61. The single-chain chimeric polypeptide of embodiment B60, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 5.

Embodiment B62. The single-chain chimeric polypeptide of embodiment B21, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 6.

Embodiment B63. The single-chain chimeric polypeptide of embodiment B62, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 6. Embodiment B64. The single-chain chimeric polypeptide of embodiment B63, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 6.

Embodiment B65. The single-chain chimeric polypeptide of embodiment B64, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 6. Embodiment B66. The single-chain chimeric polypeptide of embodiment B 17, wherein the soluble human tissue factor does not initiate coagulation in a mammal.

Embodiment Cl. A multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains.

Embodiment C2. The multi-chain chimeric polypeptide of embodiment Cl, wherein the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide.

Embodiment C3. The multi-chain chimeric polypeptide of embodiment Cl, wherein the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment C4. The multi-chain chimeric polypeptide of any one of

embodiments C1-C3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide.

Embodiment C5. The multi-chain chimeric polypeptide of any one of

embodiments C1-C3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment C6. The multi-chain chimeric polypeptide of any one of embodiments C1-C5, wherein the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide.

Embodiment C7. The multi-chain chimeric polypeptide of any one of embodiments C1-C5, wherein second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

Embodiment C8. The multi-chain chimeric polypeptide of any one of embodiments C1-C7, wherein the first target-binding domain and the second target- binding domain bind specifically to the same antigen. Embodiment C9. The multi-chain chimeric polypeptide of embodiment C8, wherein the first target-binding domain and the second target-binding domain bind specifically to the same epitope.

Embodiment C10. The multi-chain chimeric polypeptide of embodiment C9, wherein the first target-binding domain and the second target-binding domain comprise the same amino acid sequence.

Embodiment Cl 1. The multi-chain chimeric polypeptide of any one of embodiments C1-C7, wherein the first target-binding domain and the second target- binding domain bind specifically to different antigens.

Embodiment C12. The multi-chain chimeric polypeptide of any one of embodiments Cl -Cl 1, wherein one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain. Embodiment C13. The multi-chain chimeric polypeptide of embodiment Cl 2, wherein the first target-binding domain and the second target-binding domain are each antigen-binding domains. Embodiment C14. The multi-chain chimeric polypeptide of embodiment C12 or

C13, wherein antigen-binding domain comprises a scFv or a single domain antibody.

Embodiment Cl 5. The multi-chain chimeric polypeptide of any one of embodiments Cl -Cl 4, wherein one or both of the first target-binding domain and the second target-binding domain bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P- cadherin, CEACAM5, a EIL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD 122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-pRII), a ligand of TGF- PRIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-l 5, a receptor for IL- 17, a receptor for IL-l 8, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.

Embodiment Cl 6. The multi-chain chimeric polypeptide of any one of embodiments Cl -Cl 4, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin or cytokine protein. Embodiment Cl 7. The multi-chain chimeric polypeptide of embodiment Cl 6, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-l 8, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein. Embodiment Cl 8. The multi-chain chimeric polypeptide of any one of embodiments Cl -Cl 4, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin receptor, a soluble cytokine receptor, or a soluble cell surface receptor. Embodiment Cl 9. The multi-chain chimeric polypeptide of embodiment Cl 8, wherein the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28. Embodiment C20. The multi-chain chimeric polypeptide of any one of embodiments Cl -Cl 9, wherein the first chimeric polypeptide further comprises one or more additional target-binding domain(s), where at least one of the one or more additional antigen-binding domain(s) is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment C21. The multi-chain chimeric polypeptide of embodiment C20, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the at least one of the one or more additional antigen binding domain(s), and/or a linker sequence between the at least one of the one or more additional antigen-binding domain(s) and the first domain of the pair of affinity domains.

Embodiment C22. The multi-chain chimeric polypeptide of any one of embodiments Cl -Cl 9, wherein the first chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide. Embodiment C23. The multi-chain chimeric polypeptide of embodiment C22, wherein at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment C24. The multi-chain chimeric polypeptide of embodiment C22, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains. Embodiment C25. The multi-chain chimeric polypeptide of embodiment C22, wherein the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment C26. The multi-chain chimeric polypeptide of embodiment C22, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first target-binding domain.

Embodiment C27. The multi-chain chimeric polypeptide of embodiment C22, wherein at least one of the one or more additional target-binding domains is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment C28. The multi-chain chimeric polypeptide of embodiment C27, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide. Embodiment C29. The multi-chain chimeric polypeptide of embodiment C27, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment C30. The multi-chain chimeric polypeptide of embodiment C27, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment C31. The multi-chain chimeric polypeptide of embodiment C27, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment C32. The multi-chain chimeric polypeptide of embodiment C27, wherein the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, directly abuts the soluble tissue factor domain and/or the first domain of the pair of affinity domains. Embodiment C33. The multi-chain chimeric polypeptide of embodiment C27, wherein the first chimeric polypeptide further comprises a linker sequence disposed (i) between the soluble tissue factor domain and the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target- binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment C34. The multi-chain chimeric polypeptide of any one of embodiments C1-C33, wherein the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment C35. The multi-chain chimeric polypeptide of embodiment C34, wherein at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment C36. The multi-chain chimeric polypeptide of embodiment C34, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment C37. The multi-chain chimeric polypeptide of embodiment C34, wherein at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide.

Embodiment C38. The multi-chain chimeric polypeptide of embodiment C34, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second target- binding domain in the second chimeric polypeptide.

Embodiment C39. The multi-chain chimeric polypeptide of any one of embodiments C20-C38, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. Embodiment C40. The multi-chain chimeric polypeptide of embodiment C39, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope.

Embodiment C41. The multi-chain chimeric polypeptide of embodiment C40, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

Embodiment C42. The multi-chain chimeric polypeptide of embodiment C39, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same antigen. Embodiment C43. The multi-chain chimeric polypeptide of embodiment C42, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same epitope.

Embodiment C44. The multi-chain chimeric polypeptide of embodiment C43, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each comprise the same amino acid sequence.

Embodiment C45. The multi-chain chimeric polypeptide of any one of embodiments C20-C38, wherein the first target-binding domain, the second target- binding domain, and the one or more additional target-binding domains bind specifically to different antigens.

Embodiment C46. The multi-chain chimeric polypeptide of any one of embodiments C20-C45, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen binding domain. Embodiment C47. The multi-chain chimeric polypeptide of embodiment C46, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen-binding domain. Embodiment C48. The multi-chain chimeric polypeptide of embodiment C47, wherein antigen-binding domain comprises a scFv.

Embodiment C49. The multi-chain chimeric polypeptide of any one of embodiments C20-C48, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD 19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-PRII), a ligand of TGF-PRIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD3, and a receptor for CD28.

Embodiment C50. The multi-chain chimeric polypeptide of any one of embodiments C20-C48, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin, a soluble cytokine protein, or a soluble cell surface protein. Embodiment C51. The multi-chain chimeric polypeptide of embodiment C50, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-l 8, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment C52. The multi-chain chimeric polypeptide of any one of embodiments C20-C48, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin receptor, a soluble cytokine receptor, or a soluble cell surface receptor.

Embodiment C53. The multi-chain chimeric polypeptide of embodiment C52, wherein the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF- b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28.

Embodiment C54. The multi-chain chimeric polypeptide of any one of embodiments C1-C53, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide.

Embodiment C55. The multi-chain chimeric polypeptide of any one of embodiments C1-C53, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment C56. The multi-chain chimeric polypeptide of any one of embodiments C1-C55, wherein the soluble tissue factor domain is a soluble human tissue factor domain. Embodiment C57. The multi-chain chimeric polypeptide of embodiment C56, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1.

Embodiment C58. The multi-chain chimeric polypeptide of embodiment C57, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment C59. The multi-chain chimeric polypeptide of embodiment C58, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1.

Embodiment C60. The multi-chain chimeric polypeptide of any one of embodiments C56-C59, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. Embodiment C61. The multi-chain chimeric polypeptide of embodiment C60, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment C62. The multi-chain chimeric polypeptide of any one of embodiments C1-C61, wherein the soluble tissue factor domain is not capable of binding to Factor Vila.

Embodiment C63. The multi-chain chimeric polypeptide of any one of embodiments C1-C62, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment C64. The multi-chain chimeric polypeptide of any one of embodiments C1-C63, wherein the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal. Embodiment C65. The multi-chain chimeric polypeptide of any one of embodiments C1-C64, wherein the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (ILl5Ra) and a soluble IL-15.

Embodiment C66. The multi-chain chimeric polypeptide of embodiment C65, wherein the soluble IL15 has a D8N or D8A amino acid substitution.

Embodiment C67. The multi-chain chimeric polypeptide of embodiment C65 or C66, wherein the human ILl5Ra is a mature full-length ILl5Ra.

Embodiment C68. The multi-chain chimeric polypeptide of any one of embodiments C1-C64, wherein the pair of affinity domains is selected from the group consisting of: bamase and bamstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. Embodiment C69. The multi-chain chimeric polypeptide of any one of embodiments C1-C68, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminal end.

Embodiment C70. A composition comprising any of the multi-chain chimeric polypeptides of embodiments C1-C69.

Embodiment C71. The composition of embodiment C70, wherein the composition is a pharmaceutical composition. Embodiment C72. A kit comprising at least one dose of the composition of embodiment C70 or C71.

Embodiment C73. Nucleic acid encoding any of the multi-chain chimeric polypeptides of any one of embodiments C1-C69.

Embodiment C74. A vector comprising the nucleic acid of embodiment C73. Embodiment C75. The vector of embodiment C74, wherein the vector is an expression vector.

Embodiment C76. A cell comprising the nucleic acid of embodiment C73 or the vector of embodiment C74 or C75.

Embodiment C77. A method of producing a multi-chain chimeric polypeptide, the method comprising:

culturing the cell of embodiment C76 in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cell and/or the culture medium.

Embodiment C78. A multi-chain chimeric polypeptide produced by the method of embodiment C77.

Embodiment C79. The multi-chain chimeric polypeptide of embodiment C56, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 5.

Embodiment C80. The multi-chain chimeric polypeptide of embodiment C79, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5.

Embodiment C81. The multi-chain chimeric polypeptide of embodiment C80, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 5.

Embodiment C82. The multi-chain chimeric polypeptide of embodiment C81, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 5. Embodiment C83. The multi-chain chimeric polypeptide of embodiment C56, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 6.

Embodiment C84. The multi-chain chimeric polypeptide of embodiment C83, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 6.

Embodiment C85. The multi-chain chimeric polypeptide of embodiment C84, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 6. Embodiment C86. The multi-chain chimeric polypeptide of embodiment C85, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 6.

Embodiment Dl. A multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains;

the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor of IL-18 or a receptor of IL-12.

Embodiment D2. The multi-chain chimeric polypeptide of embodiment Dl, wherein the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. Embodiment D3. The multi-chain chimeric polypeptide of embodiment Dl, wherein the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide. Embodiment D4. The multi-chain chimeric polypeptide of any one of

embodiments D1-D3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide.

Embodiment D5. The multi-chain chimeric polypeptide of any one of

embodiments D1-D3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment D6. The multi-chain chimeric polypeptide of any one of embodiments D1-D5, wherein the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide.

Embodiment D7. The multi-chain chimeric polypeptide of any one of embodiments D1-D5, wherein second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

Embodiment D8. The multi-chain chimeric polypeptide of any one of embodiments D1-D7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment D9. The multi-chain chimeric polypeptide of embodiment D8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1.

Embodiment D10. The multi-chain chimeric polypeptide of embodiment D9, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment Dl l. The multi-chain chimeric polypeptide of embodiment D10, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1. Embodiment D12. The multi-chain chimeric polypeptide of any one of embodiments D8-D11, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment D13. The multi-chain chimeric polypeptide of embodiment D12, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment D14. The multi-chain chimeric polypeptide of any one of embodiments D1-D13, wherein the soluble tissue factor domain is not capable of binding to Factor Vila. Embodiment D15. The multi-chain chimeric polypeptide of any one of embodiments D1-D14, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment D16. The multi-chain chimeric polypeptide of any one of embodiments D1-D15, wherein the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

Embodiment D17. The multi-chain chimeric polypeptide of any one of embodiments D1-D16, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide.

Embodiment D18. The multi-chain chimeric polypeptide of any one of embodiments D1-D17, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment D19. The multi-chain chimeric polypeptide of any one of embodiments D1-D18, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminal end. Embodiment D20. The multi-chain chimeric polypeptide of embodiment D19, wherein the signal sequence comprises SEQ ID NO: 62.

Embodiment D21. The multi-chain chimeric polypeptide of embodiment D20, wherein the signal sequence is SEQ ID NO: 62.

Embodiment D22. The multi-chain chimeric polypeptide of any one of embodiments D1-D21, wherein the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (ILl5Ra) and a soluble IL-15.

Embodiment D23. The multi-chain chimeric polypeptide of embodiment D22, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment D24. The multi-chain chimeric polypeptide of embodiment D22, wherein the soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO: 39.

Embodiment D25. The multi-chain chimeric polypeptide of embodiment D24, wherein the soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO: 39. Embodiment D26. The multi-chain chimeric polypeptide of embodiment D25, wherein the soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO: 39.

Embodiment D27. The multi-chain chimeric polypeptide of embodiment D26, wherein the soluble IL-15 comprises SEQ ID NO: 39.

Embodiment D28. The multi-chain chimeric polypeptide of any one of embodiments D22-D27, wherein the sushi domain of ILl5Ra comprises a sushi domain from human ILl5Ra. Embodiment D29. The multi-chain chimeric polypeptide of embodiment D28, wherein the sushi domain from human ILl5Ra comprises a sequence that is 80% identical to SEQ ID NO: 10. Embodiment D30. The multi-chain chimeric polypeptide of embodiment D29, wherein the sushi domain from human ILl5Ra comprises a sequence that is 90% identical to SEQ ID NO: 10.

Embodiment D31. The multi-chain chimeric polypeptide of embodiment D30, wherein the sushi domain from human ILl5Ra comprises a sequence that is 95% identical to SEQ ID NO: 10.

Embodiment D32. The multi-chain chimeric polypeptide of embodiment D31, wherein the sushi domain from human ILl5Ra comprises SEQ ID NO: 10.

Embodiment D33. The multi-chain chimeric polypeptide of embodiment D28, wherein the sushi domain from human ILl5Ra is a mature full-length ILl5Ra.

Embodiment D34. The multi-chain chimeric polypeptide of any one of embodiments D1-D21, wherein the pair of affinity domains is selected from the group consisting of: bamase and bamstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. Embodiment D35. The multi-chain chimeric polypeptide of any one of embodiments D1-D34, wherein one or both of the first target-binding domain and the second target-binding domain is an agonistic antigen-binding domain.

Embodiment D36. The multi-chain chimeric polypeptide of embodiment D35, wherein the first target-binding domain and the second target-binding domain are each agonistic antigen-binding domains. Embodiment D37. The multi-chain chimeric polypeptide of embodiment D35 or D36, wherein antigen-binding domain comprises a scFv or single-domain antibody.

Embodiment D38. The multi-chain chimeric polypeptide of any one of embodiments D1-D34, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble IL-15 or a soluble IL-18.

Embodiment D39. The multi-chain chimeric polypeptide of embodiment D38, wherein the first target-binding domain and the second target-binding domain are each independently a soluble IL-15 or a soluble IL-18.

Embodiment D40. The multi-chain chimeric polypeptide of any one of embodiments D1-D39, wherein the first target-binding domain and the second target- binding domain both bind specifically to a receptor of IL-18 or a receptor of IL-12.

Embodiment D41. The multi-chain chimeric polypeptide of embodiment D40, wherein the first target-binding domain and the second target-binding domain bind specifically to the same epitope. Embodiment D42. The multi-chain chimeric polypeptide of embodiment D41, wherein the first target-binding domain and the second target-binding domain comprise the same amino acid sequence.

Embodiment D43. The multi-chain chimeric polypeptide of any one of embodiments D1-D39, wherein the first target-binding domain binds specifically to a receptor for IL-12, and the second target-binding domain binds specifically to a receptor for IL-18.

Embodiment D44. The multi-chain chimeric polypeptide of any one of embodiments D1-D39, wherein the first target-binding domain binds specifically to a receptor for IL-18, and the second target-binding domain bind specifically to a receptor for IL-12.

Embodiment D45. The multi-chain chimeric polypeptide of embodiment D44, wherein the first target-binding domain comprises a soluble IL-18.

Embodiment D46. The multi-chain chimeric polypeptide of embodiment D45, wherein the soluble IL-18 is a soluble human IL-18. Embodiment D47. The multi-chain chimeric polypeptide of embodiment D46, wherein the soluble human IL-18 comprises a sequence at least 80% identical to SEQ ID NO: 41.

Embodiment D48. The multi-chain chimeric polypeptide of embodiment D47, wherein the soluble human IL-18 comprises a sequence at least 90% identical to SEQ ID NO: 41.

Embodiment D49. The multi-chain chimeric polypeptide of embodiment D48, wherein the soluble human IL-18 comprises a sequence at least 95% identical to SEQ ID NO: 41.

Embodiment D50. The multi-chain chimeric polypeptide of embodiment D49, wherein the soluble human IL-18 comprises a sequence of SEQ ID NO: 41. Embodiment D51. The multi-chain chimeric polypeptide of any one of embodiments D44-D50, wherein the second target-binding domain comprises a soluble IL-12.

Embodiment D52. The multi-chain chimeric polypeptide of embodiment D51, wherein the soluble IL-18 is a soluble human IL-12. Embodiment D53. The multi-chain chimeric polypeptide of embodiment D52, wherein the soluble human IL-15 comprises a sequence of soluble human IL-12P (p40) and a sequence of soluble human IL-l2a (p35). Embodiment D54. The multi-chain chimeric polypeptide of embodiment D53, wherein the soluble human IL-15 further comprises a linker sequence between the sequence of soluble IL-12 (p40) and the sequence of soluble human IL-l2a (p35).

Embodiment D55. The multi-chain chimeric polypeptide of embodiment D54, wherein the linker sequence comprises SEQ ID NO: 7.

Embodiment D56. The multi-chain chimeric polypeptide of any one of embodiments D53-D55, wherein the sequence of soluble human IL-12P (p40) comprises a sequence that is at least 80% identical to SEQ ID NO: 33.

Embodiment D57. The multi-chain chimeric polypeptide of embodiment D56, wherein the sequence of soluble human IL-12P (p40) comprises a sequence that is at least 90% identical to SEQ ID NO: 33. Embodiment D58. The multi-chain chimeric polypeptide of embodiment D57, wherein the sequence of soluble human IL-12P (p40) comprises a sequence that is at least 95% identical to SEQ ID NO: 33.

Embodiment D59. The multi-chain chimeric polypeptide of embodiment D58, wherein the sequence of soluble human IL-12P (p40) comprises SEQ ID NO: 33.

Embodiment D60. The multi-chain chimeric polypeptide of any one of embodiments D53-D59, wherein the sequence of soluble human IL-l2a (p35) comprises a sequence that is at least 80% identical to SEQ ID NO: 35. Embodiment D61. The multi-chain chimeric polypeptide of embodiment D60, wherein the sequence of soluble human IL-l2a (p35) comprises a sequence that is at least 90% identical to SEQ ID NO: 35. Embodiment D62. The mule-chain chimeric polypeptide of embodiment D61, wherein the sequence of soluble human IL-l2a (p35) comprises a sequence that is at least 95% identical to SEQ ID NO: 35.

Embodiment D63. The multi-chain chimeric polypeptide of embodiment D62, wherein the sequence of soluble human IL-l2a (p35) comprises SEQ ID NO: 35.

Embodiment D64. The multi-chain chimeric polypeptide of embodiment Dl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 116.

Embodiment D65. The multi-chain chimeric polypeptide of embodiment D64, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 116. Embodiment D66. The multi-chain chimeric polypeptide of embodiment D65, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 116.

Embodiment D67. The multi-chain chimeric polypeptide of embodiment D66, wherein the first chimeric polypeptide comprises SEQ ID NO: 116.

Embodiment D68. The multi-chain chimeric polypeptide of embodiment D67, wherein the first chimeric polypeptide comprises SEQ ID NO: 118. Embodiment D69. The multi-chain chimeric polypeptide of any one of embodiments Dl and D64-D68, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 120. Embodiment D70. The multi-chain chimeric polypeptide of embodiment D69, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 120.

Embodiment D71. The multi-chain chimeric polypeptide of embodiment D70, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 120.

Embodiment D72. The multi-chain chimeric polypeptide of embodiment D71, wherein the second chimeric polypeptide comprises SEQ ID NO: 120.

Embodiment D73. The multi-chain chimeric polypeptide of embodiment D72, wherein the second chimeric polypeptide comprises SEQ ID NO: 122.

Embodiment D74. The multi-chain chimeric polypeptide of any one of embodiments D1-D63, wherein the first chimeric polypeptide further comprises one or more additional target-binding domain(s), where at least one of the one or more additional antigen-binding domain(s) is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains. Embodiment D75. The multi-chain chimeric polypeptide of embodiment D74, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the at least one of the one or more additional antigen binding domain(s), and/or a linker sequence between the at least one of the one or more additional antigen-binding domain(s)and the first domain of the pair of affinity domains Embodiment D76. The multi-chain chimeric polypeptide of any one of embodiments D1-D63, wherein the first chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide.

Embodiment D77. The multi-chain chimeric polypeptide of embodiment D76, wherein at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment D78. The multi-chain chimeric polypeptide of embodiment D76, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains. Embodiment D79. The multi-chain chimeric polypeptide of embodiment D76, wherein the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment D80. The multi-chain chimeric polypeptide of embodiment D76, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first target-binding domain.

Embodiment D81. The multi-chain chimeric polypeptide of embodiment D76, wherein at least one of the one or more additional target-binding domains is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment D82. The multi-chain chimeric polypeptide of embodiment D81, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment D83. The multi-chain chimeric polypeptide of embodiment D81, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment D84. The multi-chain chimeric polypeptide of embodiment D81, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment D85. The multi-chain chimeric polypeptide of embodiment D81, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment D86. The multi-chain chimeric polypeptide of embodiment D81, wherein the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, directly abuts the soluble tissue factor domain and/or the first domain of the pair of affinity domains. Embodiment D87. The multi-chain chimeric polypeptide of embodiment D81, wherein the first chimeric polypeptide further comprises a linker sequence disposed (i) between the soluble tissue factor domain and the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target- binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains. Embodiment D88. The multi-chain chimeric polypeptide of any one of embodiments D1-D63 and D74-D87, wherein the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C- terminal end of the second chimeric polypeptide. Embodiment D89. The multi-chain chimeric polypeptide of embodiment D88, wherein at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment D90. The multi-chain chimeric polypeptide of embodiment D88, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment D91. The multi-chain chimeric polypeptide of embodiment D88, wherein at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide.

Embodiment D92. The multi-chain chimeric polypeptide of embodiment D88, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second target- binding domain in the second chimeric polypeptide. Embodiment D93. The multi-chain chimeric polypeptide of any one of embodiments D74-D92, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen.

Embodiment D94. The multi-chain chimeric polypeptide of embodiment D93, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope.

Embodiment D95. The multi-chain chimeric polypeptide of embodiment D94, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

Embodiment D96. The multi-chain chimeric polypeptide of any one of embodiments D74-D92, wherein the first target-binding domain, the second target- binding domain, and the one or more additional target-binding domains bind specifically to different antigens.

Embodiment D97. The multi-chain chimeric polypeptide of any one of embodiments D74-D96, wherein the one or more additional antigen-binding domains bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD 19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CE AC AM5, a UL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-PRII), a ligand of TGF-PRIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, and a receptor for CD28.

Embodiment D98. The multi-chain chimeric polypeptide of any one of embodiments D74-D96, wherein the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein .

Embodiment D99. The multi-chain chimeric polypeptide of embodiment D98, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-l 7, IL- 18, IL-21 , PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP 16-binding protein.

Embodiment D100. The multi-chain chimeric polypeptide of any one of embodiments D74-D96, wherein the one or more additional target-binding domains is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor.

Embodiment D101. The multi-chain chimeric polypeptide of embodiment D100, wherein the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF- bIIPI, a soluble NKG2D, a soluble NKP30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, or a soluble CD28.

Embodiment D102. A composition comprising any of the multi-chain chimeric polypeptides of embodiments D1-D101. Embodiment D103. The composition of embodiment D102, wherein the composition is a pharmaceutical composition.

Embodiment D104. A kit comprising at least one dose of the composition of embodiment D 102 or D 103.

Embodiment D105. Nucleic acid encoding any of the multi-chain chimeric polypeptides of any one of embodiments D1-D101. Embodiment D106. A vector comprising the nucleic acid of embodiment D 105.

Embodiment D107. The vector of embodiment D 106, wherein the vector is an expression vector. Embodiment D108. A cell comprising the nucleic acid of embodiment D 105 or the vector of embodiment D 106 or D 107.

Embodiment D109. A method of producing a multi-chain chimeric polypeptide, the method comprising:

culturing the cell of embodiment D 108 in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cell and/or the culture medium. Embodiment Dl 10. A multi-chain chimeric polypeptide produced by the method of embodiment D109.

Embodiment Dlll. The multi-chain chimeric polypeptide of embodiment D8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 5. Embodiment D112. The multi-chain chimeric polypeptide of embodiment Dll l, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5.

Embodiment D113. The multi-chain chimeric polypeptide of embodiment D112, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 5.

Embodiment D114. The multi-chain chimeric polypeptide of embodiment D113, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 5.

Embodiment Dll 5. The multi-chain chimeric polypeptide of embodiment D8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 6.

Embodiment D116. The multi-chain chimeric polypeptide of embodiment Dll 5, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 6.

Embodiment D117. The multi-chain chimeric polypeptide of embodiment D116, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 6.

Embodiment Dl 18. The multi-chain chimeric polypeptide of embodiment Dl 17, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 6. Embodiment El. A multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and

the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor of IL-21 or a ligand of tumor growth factor receptor b II (TGFpRII).

Embodiment E2. The multi-chain chimeric polypeptide of embodiment El, wherein the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide.

Embodiment E3. The multi-chain chimeric polypeptide of embodiments El, wherein the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment E4. The multi-chain chimeric polypeptide of any one of

embodiments E1-E3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. Embodiment E5. The multi-chain chimeric polypeptide of any one of

embodiments E1-E3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment E6. The multi-chain chimeric polypeptide of any one of embodiments E1-E5, wherein the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide.

Embodiment E7. The multi-chain chimeric polypeptide of any one of embodiments E1-E5, wherein second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

Embodiment E8. The multi-chain chimeric polypeptide of any one of embodiments E1-E7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment E9. The multi-chain chimeric polypeptide of embodiment E8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1.

Embodiment E10. The multi-chain chimeric polypeptide of embodiment E9, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment El l. The multi-chain chimeric polypeptide of embodiment E10, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1. Embodiment E12. The multi-chain chimeric polypeptide of any one of embodiments E8-E11, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment E13. The multi-chain chimeric polypeptide of embodiment E12, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment E14. The multi-chain chimeric polypeptide of any one of embodiments E1-E13, wherein the soluble tissue factor domain is not capable of binding to Factor Vila. Embodiment El 5. The multi-chain chimeric polypeptide of any one of embodiments E1-E14, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment El 6. The multi-chain chimeric polypeptide of any one of embodiments E1-E15, wherein the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

Embodiment El 7. The multi-chain chimeric polypeptide of any one of embodiments E1-E16, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide.

Embodiment El 8. The multi-chain chimeric polypeptide of any one of embodiments E1-E17, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment El 9. The multi-chain chimeric polypeptide of any one of embodiments E1-E18, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminal end. Embodiment E20. The multi-chain chimeric polypeptide of embodiment E19, wherein the signal sequence comprises SEQ ID NO: 62.

Embodiment E21. The multi-chain chimeric polypeptide of embodiment E20, wherein the signal sequence is SEQ ID NO: 62.

Embodiment E22. The multi-chain chimeric polypeptide of any one of embodiments E1-E21, wherein the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (ILl5Ra and a soluble IL-15.

Embodiment E23. The multi-chain chimeric polypeptide of embodiment E22, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment E24. The multi-chain chimeric polypeptide of embodiment E22, wherein the soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO: 39.

Embodiment E25. The multi-chain chimeric polypeptide of embodiment E24, wherein the soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO: 39. Embodiment E26. The multi-chain chimeric polypeptide of embodiment E25, wherein the soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO: 39.

Embodiment E27. The multi-chain chimeric polypeptide of embodiment E26, wherein the soluble IL-15 comprises SEQ ID NO: 39.

Embodiment E28. The multi-chain chimeric polypeptide of any one of embodiments E22-E27, wherein the sushi domain of ILl5Ra comprises a sushi domain from human ILl5Ra. Embodiment E29. The multi-chain chimeric polypeptide of embodiment E28, wherein the sushi domain from human ILl5Ra comprises a sequence that is 80% identical to SEQ ID NO: 10. Embodiment E30. The multi-chain chimeric polypeptide of embodiment E29, wherein the sushi domain from human ILl5Ra comprises a sequence that is 90% identical to SEQ ID NO: 10.

Embodiment E31. The multi-chain chimeric polypeptide of embodiment E30, wherein the sushi domain from human ILl5Ra comprises a sequence that is 95% identical to SEQ ID NO: 10.

Embodiment E32. The multi-chain chimeric polypeptide of embodiment E31, wherein the sushi domain from human ILl5Ra comprises SEQ ID NO: 10.

Embodiment E33. The multi-chain chimeric polypeptide of embodiment E28, wherein the sushi domain from human ILl5Ra is a mature full-length ILl5Ra.

Embodiment E34. The multi-chain chimeric polypeptide of any one of embodiments E1-E21, wherein the pair of affinity domains is selected from the group consisting of: bamase and bamstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. Embodiment E35. The multi-chain chimeric polypeptide of any one of embodiments E1-E34, wherein one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain.

Embodiment E36. The multi-chain chimeric polypeptide of embodiment E35, wherein the first target-binding domain and the second target-binding domain are antigen-binding domains. Embodiment E37. The multi-chain chimeric polypeptide of embodiment E35 or E36, wherein antigen-binding domain comprises a scFv or single-domain antibody.

Embodiment E38. The multi-chain chimeric polypeptide of any one of embodiments E1-E34, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble IL-21 or a soluble TGFpRII.

Embodiment E39. The multi-chain chimeric polypeptide of any one of embodiments E1-E38, wherein the first target-binding domain and the second target- binding domain both bind specifically to a receptor of IL-21 or a ligand of TGFpRII.

Embodiment E40. The multi-chain chimeric polypeptide of embodiment E39, wherein the first target-binding domain and the second target-binding domain bind specifically to the same epitope.

Embodiment E41. The multi-chain chimeric polypeptide of embodiment E40, wherein the first target-binding domain and the second target-binding domain comprise the same amino acid sequence. Embodiment E42. The multi-chain chimeric polypeptide of any one of embodiments E1-E38, wherein the first target-binding domain binds specifically to a ligand of TGFpRII, and the second target-binding domain binds specifically to a receptor for IL-21. Embodiment E43. The multi-chain chimeric polypeptide of any one of embodiments E1-E38, wherein the first target-binding domain binds specifically to a receptor for IL-21, and the second target-binding domain bind specifically to a ligand of TGFpRII. Embodiment E44. The multi-chain chimeric polypeptide of embodiment E43, wherein the first target-binding domain comprises a soluble IL-21. Embodiment E45. The multi-chain chimeric polypeptide of embodiment E44, wherein the soluble IL-21 is a soluble human IL-21.

Embodiment E46. The multi-chain chimeric polypeptide of embodiment E45, wherein the soluble human IL-21 comprises a sequence at least 80% identical to SEQ ID NO: 124.

Embodiment E47. The multi-chain chimeric polypeptide of embodiment E46, wherein the soluble human IL-21 comprises a sequence at least 90% identical to SEQ ID NO: 124.

Embodiment E48. The multi-chain chimeric polypeptide of embodiment E47, wherein the soluble human IL-21 comprises a sequence at least 95% identical to SEQ ID NO: 124.

Embodiment E49. The multi-chain chimeric polypeptide of embodiment E48, wherein the soluble human IL-21 comprises a sequence of SEQ ID NO: 124.

Embodiment E50. The multi-chain chimeric polypeptide of any one of embodiments E43-E49, wherein the second target-binding domain comprises a soluble TGFpRII.

Embodiment E51. The multi-chain chimeric polypeptide of embodiment E50, wherein the soluble TGFpRII is a soluble human TGFpRII.

Embodiment E52. The multi-chain chimeric polypeptide of embodiment E51, wherein the soluble human TGFpRII comprises a first sequence of soluble human TGFpRII and a second sequence of soluble human TGFpRII. Embodiment E53. The multi-chain chimeric polypeptide of embodiment E52, wherein the soluble human TGFpRII further comprises a linker sequence between the first sequence of soluble human TGFpRII and the second sequence of soluble human TGFpRII.

Embodiment E54. The multi-chain chimeric polypeptide of embodiment E53, wherein the linker sequence comprises SEQ ID NO: 7.

Embodiment E55. The multi-chain chimeric polypeptide of any one of embodiments E52-E54, wherein the first sequence of soluble human TGFpRII comprises a sequence that is at least 80% identical to SEQ ID NO: 56.

Embodiment E56. The multi-chain chimeric polypeptide of embodiment E55, wherein the first sequence of soluble human TGFpRII comprises a sequence that is at least 90% identical to SEQ ID NO: 56. Embodiment E57. The multi-chain chimeric polypeptide of embodiment E56, wherein the first sequence of soluble human TGFpRII comprises a sequence that is at least 95% identical to SEQ ID NO: 56.

Embodiment E58. The multi-chain chimeric polypeptide of embodiment E57, wherein the first sequence of soluble human TGFpRII comprises SEQ ID NO: 56.

Embodiment E59. The multi-chain chimeric polypeptide of any one of embodiments E52-E58, wherein the second sequence of soluble human TGFpRII comprises a sequence that is at least 80% identical to SEQ ID NO: 56.

Embodiment E60. The multi-chain chimeric polypeptide of embodiment E59, wherein the second sequence of soluble human TGFpRII comprises a sequence that is at least 90% identical to SEQ ID NO: 56. Embodiment E61. The mule-chain chimeric polypeptide of embodiment E60, wherein the second sequence of soluble human TGFpRII comprises a sequence that is at least 95% identical to SEQ ID NO: 56. Embodiment E62. The multi-chain chimeric polypeptide of embodiment E61, wherein the second sequence of soluble human TGFpRII comprises SEQ ID NO: 56.

Embodiment E63. The multi-chain chimeric polypeptide of embodiment El, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 126.

Embodiment E64. The multi-chain chimeric polypeptide of embodiment E63, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 126.

Embodiment E65. The multi-chain chimeric polypeptide of embodiment E64, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 126. Embodiment E66. The multi-chain chimeric polypeptide of embodiment E65, wherein the first chimeric polypeptide comprises SEQ ID NO: 126.

Embodiment E67. The multi-chain chimeric polypeptide of embodiment E66, wherein the first chimeric polypeptide comprises SEQ ID NO: 128.

Embodiment E68. The multi-chain chimeric polypeptide of any one of embodiments El and E63-E67, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 130. Embodiment E69. The multi-chain chimeric polypeptide of embodiment E68, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 130. Embodiment E70. The multi-chain chimeric polypeptide of embodiment E69, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 130.

Embodiment E71. The multi-chain chimeric polypeptide of embodiment E70, wherein the second chimeric polypeptide comprises SEQ ID NO: 130.

Embodiment E72. The multi-chain chimeric polypeptide of embodiment E71, wherein the second chimeric polypeptide comprises SEQ ID NO: 132. Embodiment E73. The multi-chain chimeric polypeptide of any one of embodiments E1-E62, wherein the first chimeric polypeptide further comprises one or more additional target-binding domain(s), where at least one of the one or more additional antigen-binding domain(s) is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment E74. The multi-chain chimeric polypeptide of embodiment E73, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the at least one of the one or more additional antigen binding domain(s), and/or a linker sequence between the at least one of the one or more additional antigen-binding domain(s) and the first domain of the pair of affinity domains.

Embodiment E75. The multi-chain chimeric polypeptide of any one of embodiments E1-E62, wherein the first chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide. Embodiment E76. The multi-chain chimeric polypeptide of embodiment E75, wherein at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment E77. The multi-chain chimeric polypeptide of embodiment E75, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains. Embodiment E78. The multi-chain chimeric polypeptide of embodiment E75, wherein the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment E79. The multi-chain chimeric polypeptide of embodiment E75, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first target-binding domain.

Embodiment E80. The multi-chain chimeric polypeptide of embodiment E75, wherein at least one of the one or more additional target-binding domains is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment E81. The multi-chain chimeric polypeptide of embodiment E80, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide. Embodiment E82. The multi-chain chimeric polypeptide of embodiment E80, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment E83. The multi-chain chimeric polypeptide of embodiment E80, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment E84. The multi-chain chimeric polypeptide of embodiment E80, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment E85. The multi-chain chimeric polypeptide of embodiment E80, wherein the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, directly abuts the soluble tissue factor domain and/or the first domain of the pair of affinity domains. Embodiment E86. The multi-chain chimeric polypeptide of embodiment E80, wherein the first chimeric polypeptide further comprises a linker sequence disposed (i) between the soluble tissue factor domain and the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target- binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment E87. The multi-chain chimeric polypeptide of any one of embodiments E1-E62 and E73-E86, wherein the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C- terminal end of the second chimeric polypeptide.

Embodiment E88. The multi-chain chimeric polypeptide of embodiment E87, wherein at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment E89. The multi-chain chimeric polypeptide of embodiment E87, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment E90. The multi-chain chimeric polypeptide of embodiment E87, wherein at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide.

Embodiment E91. The multi-chain chimeric polypeptide of embodiment E87, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second target- binding domain in the second chimeric polypeptide.

Embodiment E92. The multi-chain chimeric polypeptide of any one of embodiments E73-E91, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. Embodiment E93. The multi-chain chimeric polypeptide of embodiment E92, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope.

Embodiment E94. The multi-chain chimeric polypeptide of embodiment E93, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

Embodiment E95. The multi-chain chimeric polypeptide of any one of embodiments E73-E91, wherein the first target-binding domain, the second target- binding domain, and the one or more additional target-binding domains bind specifically to different antigens.

Embodiment E96. The multi-chain chimeric polypeptide of any one of embodiments E73-E95, wherein the one or more additional antigen-binding domains bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD 19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CE AC AM5, a UL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-D, a ligand of TGF-b receptor II (TGF-PRII), a ligand of TGF-PRIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULPl6-binding protein, a receptor for CD155, and a receptor for CD28. Embodiment E97. The multi-chain chimeric polypeptide of any one of embodiments E73-E95, wherein the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. Embodiment E98. The multi-chain chimeric polypeptide of embodiment E97, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-l 8, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment E99. The multi-chain chimeric polypeptide of any one of embodiments E73-E95, wherein the one or more additional target-binding domains is a soluble interleukin or cytokine receptor. Embodiment El 00. The multi-chain chimeric polypeptide of embodiment E99, wherein the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, , or a soluble CD28. Embodiment E101. A composition comprising any of the multi-chain chimeric polypeptides of embodiments E1-E100.

Embodiment E102. The composition of embodiment E101, wherein the composition is a pharmaceutical composition.

Embodiment El 03. A kit comprising at least one dose of the composition of embodiment El 01 or El02.

Embodiment El 04. Nucleic acid encoding any of the multi-chain chimeric polypeptides of any one of embodiments E1-E100. Embodiment El 05. A vector comprising the nucleic acid of embodiment El 04.

Embodiment E106. The vector of embodiment E105, wherein the vector is an expression vector.

Embodiment El 07. A cell comprising the nucleic acid of embodiment C161 or the vector of embodiment E105 or E106.

Embodiment E108. A method of producing a multi-chain chimeric polypeptide, the method comprising:

culturing the cell of embodiment El 07 in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cell and/or the culture medium.

Embodiment El 09. A multi-chain chimeric polypeptide produced by the method of embodiment E108.

Embodiment El 10. The multi-chain chimeric polypeptide of embodiment E12, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 5.

Embodiment El 11. The multi-chain chimeric polypeptide of embodiment El 10, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5.

Embodiment El 12. The multi-chain chimeric polypeptide of embodiment El 11, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 5. Embodiment El 13. The multi-chain chimeric polypeptide of embodiment El 12, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 5. Embodiment El 14. The multi-chain chimeric polypeptide of embodiment E12, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 6.

Embodiment El 15. The multi-chain chimeric polypeptide of embodiment El 14, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 6.

Embodiment El 16. The multi-chain chimeric polypeptide of embodiment El 15, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 6.

Embodiment El 17. The multi-chain chimeric polypeptide of embodiment El 16, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 6.

Embodiment Fl. A multi-chain chimeric polypeptide comprising:

(c) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a first domain of a pair of affinity domains;

(d) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains;

the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor of IL-21 or a receptor of IL-7.

Embodiment F2. The multi-chain chimeric polypeptide of embodiment Fl, wherein the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. Embodiment F3. The multi-chain chimeric polypeptide of embodiment Fl, wherein the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide. Embodiment F4. The multi-chain chimeric polypeptide of any one of

embodiments F1-F3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide.

Embodiment F5. The multi-chain chimeric polypeptide of any one of

embodiments F1-F3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment F6. The multi-chain chimeric polypeptide of any one of embodiments F1-F5, wherein the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide.

Embodiment F7. The multi-chain chimeric polypeptide of any one of embodiments F1-F5, wherein second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

Embodiment F8. The multi-chain chimeric polypeptide of any one of embodiments F1-F7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment F9. The multi-chain chimeric polypeptide of embodiment F8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1.

Embodiment F 10. The multi-chain chimeric polypeptide of embodiment F9, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment Fl 1. The multi-chain chimeric polypeptide of embodiment F 10, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1. Embodiment F 12. The multi-chain chimeric polypeptide of embodiment Fl 1, wherein the soluble human tissue factor domain comprises SEQ ID NO: 1. Embodiment F 13. The multi-chain chimeric polypeptide of embodiment F8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 5. Embodiment F 14. The multi-chain chimeric polypeptide of embodiment F 13, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5.

Embodiment F 15. The multi-chain chimeric polypeptide of embodiment F 14, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 5.

Embodiment F 16. The multi-chain chimeric polypeptide of embodiment F 15, wherein the soluble human tissue factor domain comprises SEQ ID NO: 5.

Embodiment F 17. The multi-chain chimeric polypeptide of embodiment F8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 6. Embodiment F 18. The multi-chain chimeric polypeptide of embodiment F 17, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 6.

Embodiment F 19. The multi-chain chimeric polypeptide of embodiment F 18, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 6.

Embodiment F20. The multi-chain chimeric polypeptide of embodiment F 19, wherein the soluble human tissue factor domain comprises SEQ ID NO: 6. Embodiment F21. The multi-chain chimeric polypeptide of any one of embodiments F8-F11, F13-F15, and F17-F19, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment F22. The multi-chain chimeric polypeptide of embodiment F21, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment F23. The multi-chain chimeric polypeptide of any one of embodiments F1-F22, wherein the soluble tissue factor domain is not capable of binding to Factor Vila. Embodiment F24. The multi-chain chimeric polypeptide of any one of embodiments F1-F23, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment F25. The multi-chain chimeric polypeptide of any one of embodiments F1-F24, wherein the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

Embodiment F26. The multi-chain chimeric polypeptide of any one of embodiments F1-F25, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide.

Embodiment F27. The multi-chain chimeric polypeptide of any one of embodiments F1-F26, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment F28. The multi-chain chimeric polypeptide of any one of embodiments F1-F27, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminal end. Embodiment F29. The multi-chain chimeric polypeptide of embodiment F28, wherein the signal sequence comprises SEQ ID NO: 62.

Embodiment F30. The multi-chain chimeric polypeptide of embodiment F28, wherein the signal sequence is SEQ ID NO: 44.

Embodiment F31. The multi-chain chimeric polypeptide of any one of embodiments F1-F30, wherein the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (ILl5Ra) and a soluble IL-15.

Embodiment F32. The multi-chain chimeric polypeptide of embodiment F31, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment F33. The multi-chain chimeric polypeptide of embodiment F31, wherein the soluble IL-15 comprises a sequence that is at least 80% identical to SEQ ID NO: 39.

Embodiment F34. The multi-chain chimeric polypeptide of embodiment F33, wherein the soluble IL-15 comprises a sequence that is at least 90% identical to SEQ ID NO: 39.

Embodiment F35. The multi-chain chimeric polypeptide of embodiment F34, wherein the soluble IL-15 comprises a sequence that is at least 95% identical to SEQ ID NO: 39.

Embodiment F36. The multi-chain chimeric polypeptide of embodiment F35, wherein the soluble IL-15 comprises SEQ ID NO: 39.

Embodiment F37. The multi-chain chimeric polypeptide of any one of embodiments F31-F36, wherein the sushi domain of ILl5Ra comprises a sushi domain from human ILl5Ra. Embodiment F38. The multi-chain chimeric polypeptide of embodiment F37, wherein the sushi domain from human ILl5Ra comprises a sequence that is at least 80% identical to SEQ ID NO: 10. Embodiment F39. The multi-chain chimeric polypeptide of embodiment F38, wherein the sushi domain from human ILl5Ra comprises a sequence that is at least 90% identical to SEQ ID NO: 10.

Embodiment F40. The multi-chain chimeric polypeptide of embodiment F39, wherein the sushi domain from human ILl5Ra comprises a sequence that is at least 95% identical to SEQ ID NO: 10.

Embodiment F41. The multi-chain chimeric polypeptide of embodiment F40, wherein the sushi domain from human ILl5Ra comprises SEQ ID NO: 10.

Embodiment F42. The multi-chain chimeric polypeptide of embodiment F37, wherein the sushi domain from human ILl5Ra is a mature full-length ILl5Ra.

Embodiment F43. The multi-chain chimeric polypeptide of any one of embodiments F1-F30, wherein the pair of affinity domains is selected from the group consisting of: bamase and bamstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. Embodiment F44. The multi-chain chimeric polypeptide of any one of embodiments F1-F43, wherein one or both of the first target-binding domain and the second target-binding domain is an agonistic antigen-binding domain.

Embodiment F45. The multi-chain chimeric polypeptide of embodiment F44, wherein the first target-binding domain and the second target-binding domain are each agonistic antigen-binding domains. Embodiment F46. The multi-chain chimeric polypeptide of embodiment F44 or F45, wherein antigen-binding domain comprises a scFv or single-domain antibody.

Embodiment F47. The multi-chain chimeric polypeptide of any one of embodiments F1-F43, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble IL-21 or a soluble IL-7.

Embodiment F48. The multi-chain chimeric polypeptide of embodiment F47, wherein the first target-binding domain and the second target-binding domain are each independently a soluble IL-21 or a soluble IL-7.

Embodiment F49. The multi-chain chimeric polypeptide of any one of embodiments F1-F48, wherein the first target-binding domain and the second target binding domain both bind specifically to a receptor of IL-21 or a receptor of IL-7.

Embodiment F50. The multi-chain chimeric polypeptide of embodiment F49, wherein the first target-binding domain and the second target-binding domain bind specifically to the same epitope. Embodiment F51. The multi-chain chimeric polypeptide of embodiment F50, wherein the first target-binding domain and the second target-binding domain comprise the same amino acid sequence.

Embodiment F52. The multi-chain chimeric polypeptide of any one of embodiments F1-F48, wherein the first target-binding domain binds specifically to a receptor for IL-21, and the second target-binding domain binds specifically to a receptor for IL-7.

Embodiment F53. The multi-chain chimeric polypeptide of any one of embodiments F1-F48, wherein the first target-binding domain binds specifically to a receptor for IL-7, and the second target-binding domain bind specifically to a receptor for IL-21.

Embodiment F54. The multi-chain chimeric polypeptide of embodiment F53, wherein the first target-binding domain comprises a soluble IL-21.

Embodiment F55. The multi-chain chimeric polypeptide of embodiment F54, wherein the soluble IL-21 is a soluble human IL-21. Embodiment F56. The multi-chain chimeric polypeptide of embodiment F55, wherein the soluble human IL-21 comprises a sequence at least 80% identical to SEQ ID NO: 124.

Embodiment F57. The multi-chain chimeric polypeptide of embodiment F56, wherein the soluble human IL-21 comprises a sequence at least 90% identical to SEQ ID NO: 124.

Embodiment F58. The multi-chain chimeric polypeptide of embodiment F57, wherein the soluble human IL-21 comprises a sequence at least 95% identical to SEQ ID NO: 124.

Embodiment F59. The multi-chain chimeric polypeptide of embodiment F58, wherein the soluble human IL-21 comprises a sequence of SEQ ID NO: 124. Embodiment F60. The multi-chain chimeric polypeptide of any one of embodiments F53-F59, wherein the second target-binding domain comprises a soluble IL-7.

Embodiment F61. The multi-chain chimeric polypeptide of embodiment D60, wherein the soluble IL-7 is a soluble human IL-7. Embodiment F62. The multi-chain chimeric polypeptide of embodiment F61, wherein the soluble human IL-7 comprises a sequence at least 80% identical to SEQ ID NO: 135. Embodiment F63. The multi-chain chimeric polypeptide of embodiment F62, wherein the soluble human IL-7 comprises a sequence at least 90% identical to SEQ ID NO: 135.

Embodiment F64. The multi-chain chimeric polypeptide of embodiment F63, wherein the soluble human IL-7 comprises a sequence at least 95% identical to SEQ ID NO: 135.

Embodiment F65. The multi-chain chimeric polypeptide of embodiment F64, wherein the soluble human IL-7 comprises a sequence of SEQ ID NO: 135.

Embodiment F66. The multi-chain chimeric polypeptide of embodiment Fl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 141. Embodiment F67. The multi-chain chimeric polypeptide of embodiment F66, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 141.

Embodiment F68. The multi-chain chimeric polypeptide of embodiment F67, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 141.

Embodiment F69. The multi-chain chimeric polypeptide of embodiment F68, wherein the first chimeric polypeptide comprises SEQ ID NO: 141. Embodiment F70. The multi-chain chimeric polypeptide of embodiment F69, wherein the first chimeric polypeptide comprises SEQ ID NO: 143.

Embodiment F71. The multi-chain chimeric polypeptide of any one of embodiments Fl and F66-F70, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 145.

Embodiment F72. The multi-chain chimeric polypeptide of embodiment F71, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 145.

Embodiment F73. The multi-chain chimeric polypeptide of embodiment F72, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 145.

Embodiment F74. The multi-chain chimeric polypeptide of embodiment F73, wherein the second chimeric polypeptide comprises SEQ ID NO: 145.

Embodiment F75. The multi-chain chimeric polypeptide of embodiment F74, wherein the second chimeric polypeptide comprises SEQ ID NO: 147.

Embodiment F76. The multi-chain chimeric polypeptide of embodiment Fl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 126.

Embodiment F77. The multi-chain chimeric polypeptide of embodiment F76, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 126. Embodiment F78. The multi-chain chimeric polypeptide of embodiment F77, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 126. Embodiment F79. The multi-chain chimeric polypeptide of embodiment F68, wherein the first chimeric polypeptide comprises SEQ ID NO: 126.

Embodiment F80. The multi-chain chimeric polypeptide of embodiment F69, wherein the first chimeric polypeptide comprises SEQ ID NO: 128.

Embodiment F81. The multi-chain chimeric polypeptide of any one of embodiments Fl and F76-F80, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 137. Embodiment F82. The multi-chain chimeric polypeptide of embodiment F81, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 137.

Embodiment F83. The multi-chain chimeric polypeptide of embodiment F82, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 137.

Embodiment F84. The multi-chain chimeric polypeptide of embodiment F83, wherein the second chimeric polypeptide comprises SEQ ID NO: 137.

Embodiment F85. The multi-chain chimeric polypeptide of embodiment F84, wherein the second chimeric polypeptide comprises SEQ ID NO: 139.

Embodiment F86. The multi-chain chimeric polypeptide of any one of embodiments F1-F65, wherein the first chimeric polypeptide further comprises one or more additional target-binding domain(s), where at least one of the one or more additional antigen-binding domain(s) is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment F87. The multi-chain chimeric polypeptide of embodiment F86, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the at least one of the one or more additional antigen binding domain(s), and/or a linker sequence between the at least one of the one or more additional antigen-binding domain(s)and the first domain of the pair of affinity domains. Embodiment F88. The multi-chain chimeric polypeptide of any one of embodiments F1-F65, wherein the first chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide. Embodiment F89. The multi-chain chimeric polypeptide of embodiment F88, wherein at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment F90. The multi-chain chimeric polypeptide of embodiment F88, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains.

Embodiment F91. The multi-chain chimeric polypeptide of embodiment F88, wherein the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment F92. The multi-chain chimeric polypeptide of embodiment F88, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first target-binding domain. Embodiment F93. The multi-chain chimeric polypeptide of embodiment F88, wherein at least one of the one or more additional target-binding domains is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment F94. The multi-chain chimeric polypeptide of embodiment F93, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment F95. The multi-chain chimeric polypeptide of embodiment F93, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment F96. The multi-chain chimeric polypeptide of embodiment F93, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment F97. The multi-chain chimeric polypeptide of embodiment F93, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment F98. The multi-chain chimeric polypeptide of embodiment F93, wherein the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, directly abuts the soluble tissue factor domain and/or the first domain of the pair of affinity domains.

Embodiment F99. The multi-chain chimeric polypeptide of embodiment F93, wherein the first chimeric polypeptide further comprises a linker sequence disposed (i) between the soluble tissue factor domain and the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target- binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment F 100. The multi-chain chimeric polypeptide of any one of embodiments F1-F65 and F86-F99, wherein the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C- terminal end of the second chimeric polypeptide.

Embodiment F 101. The multi-chain chimeric polypeptide of embodiment F100, wherein at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide. Embodiment F 102. The multi-chain chimeric polypeptide of embodiment F100, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide. Embodiment F 103. The multi-chain chimeric polypeptide of embodiment F100, wherein at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide. Embodiment F 104. The multi-chain chimeric polypeptide of embodiment F100, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second target- binding domain in the second chimeric polypeptide. Embodiment F 105. The multi-chain chimeric polypeptide of any one of embodiments F86-F104, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. Embodiment F 106. The multi-chain chimeric polypeptide of embodiment F105, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope. Embodiment F 107. The multi-chain chimeric polypeptide of embodiment F106, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence. Embodiment F 108. The multi-chain chimeric polypeptide of any one of embodiments F86-F104, wherein the first target-binding domain, the second target- binding domain, and the one or more additional target-binding domains bind specifically to different antigens. Embodiment F 109. The multi-chain chimeric polypeptide of any one of embodiments F86-F108, wherein the one or more additional antigen-binding domains bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD 19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-PRII), a ligand of TGF-PRIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, and a receptor for CD28.

Embodiment Fl 10. The multi-chain chimeric polypeptide of any one of embodiments F86-F108, wherein the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein.

Embodiment Fl 11. The multi-chain chimeric polypeptide of embodiment Fl 10, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-l 7, IL-l 8, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment Fl 12. The multi-chain chimeric polypeptide of any one of embodiments F86-F108, wherein the one or more additional target-binding domains is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor. Embodiment Fl 13. The multi-chain chimeric polypeptide of embodiment Fl 12, wherein the soluble receptor is a soluble TGF-b receptor II (TGF-PRII), a soluble TGF- PRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, or a soluble CD28.

Embodiment Fl 14. A composition comprising any of the multi-chain chimeric polypeptides of embodiments Fl-Fl 13.

Embodiment Fl 15. The composition of embodiment Fl 14, wherein the composition is a pharmaceutical composition.

Embodiment Fl 16. A kit comprising at least one dose of the composition of embodiment Fl l4 or Fl l5.

Embodiment Fl 17. Nucleic acid encoding any of the multi-chain chimeric polypeptides of any one of embodiments Fl-Fl 13.

Embodiment Fl 18. A vector comprising the nucleic acid of embodiment Fl 17.

Embodiment Fl 19. The vector of embodiment Fl 18, wherein the vector is an expression vector.

Embodiment F120. A cell comprising the nucleic acid of embodiment Fl 17 or the vector of embodiment Fl 18 or Fl 19.

Embodiment F121. A method of producing a multi-chain chimeric polypeptide, the method comprising:

culturing the cell of embodiment F 120 in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide; and recovering the multi-chain chimeric polypeptide from the cell and/or the culture medium.

Embodiment F122. A multi-chain chimeric polypeptide produced by the method of embodiment F 121.

Embodiment Gl. A multi-chain chimeric polypeptide comprising:

(e) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a first domain of a pair of affinity domains;

(f) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and

the first target-binding domain and the second targeting-binding domain each independently bind specifically to: a receptor for IL-7, CD16, a receptor for IL-21, TGF- b, or a receptor for CD137L.

Embodiment G2. The multi-chain chimeric polypeptide of embodiment Gl, wherein the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide.

Embodiment G3. The multi-chain chimeric polypeptide of embodiment Gl, wherein the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment G4. The multi-chain chimeric polypeptide of any one of

embodiments G1-G3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. Embodiment G5. The multi-chain chimeric polypeptide of any one of

embodiments G1-G3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment G6. The multi-chain chimeric polypeptide of any one of embodiments G1-G5, wherein the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide.

Embodiment G7. The multi-chain chimeric polypeptide of any one of embodiments G1-G5, wherein second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

Embodiment G8. The multi-chain chimeric polypeptide of any one of embodiments G1-G7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment G9. The multi-chain chimeric polypeptide of embodiment G8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1.

Embodiment G10. The multi-chain chimeric polypeptide of embodiment G9, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment Gl 1. The multi-chain chimeric polypeptide of embodiment G10, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1. Embodiment G12. The multi-chain chimeric polypeptide of any one of embodiments G8-G11, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment G13. The multi-chain chimeric polypeptide of embodiment G12, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment G14. The multi-chain chimeric polypeptide of any one of embodiments G1-G13, wherein the soluble tissue factor domain is not capable of binding to Factor Vila. Embodiment G15. The multi-chain chimeric polypeptide of any one of embodiments G1-G14, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment G16. The multi-chain chimeric polypeptide of any one of embodiments G1-G15, wherein the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

Embodiment G17. The multi-chain chimeric polypeptide of any one of embodiments G1-G16, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide.

Embodiment G18. The multi-chain chimeric polypeptide of any one of embodiments G1-G17, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment G19. The multi-chain chimeric polypeptide of any one of embodiments G1-G18, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminal end. Embodiment G20. The multi-chain chimeric polypeptide of embodiment G19, wherein the signal sequence comprises SEQ ID NO: 62.

Embodiment G21. The multi-chain chimeric polypeptide of embodiment G20, wherein the signal sequence is SEQ ID NO: 62.

Embodiment G22. The multi-chain chimeric polypeptide of any one of embodiments G1-G21, wherein the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (ILl5Ra) and a soluble IL-15.

Embodiment G23. The multi-chain chimeric polypeptide of embodiment G22, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment G24. The multi-chain chimeric polypeptide of embodiment G22, wherein the soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO: 39.

Embodiment G25. The multi-chain chimeric polypeptide of embodiment G24, wherein the soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO: 39. Embodiment G26. The multi-chain chimeric polypeptide of embodiment G25, wherein the soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO: 39.

Embodiment G27. The multi-chain chimeric polypeptide of embodiment G26, wherein the soluble IL-15 comprises SEQ ID NO: 39.

Embodiment G28. The multi-chain chimeric polypeptide of any one of embodiments G22-G27, wherein the sushi domain of ILl5Ra comprises a sushi domain from human ILl5Ra. Embodiment G29. The multi-chain chimeric polypeptide of embodiment G28, wherein the sushi domain from human ILl5Ra comprises a sequence that is 80% identical to SEQ ID NO: 10. Embodiment G30. The multi-chain chimeric polypeptide of embodiment G29, wherein the sushi domain from human ILl5Ra comprises a sequence that is 90% identical to SEQ ID NO: 10.

Embodiment G31. The multi-chain chimeric polypeptide of embodiment G30, wherein the sushi domain from human ILl5Ra comprises a sequence that is 95% identical to SEQ ID NO: 10.

Embodiment G32. The multi-chain chimeric polypeptide of embodiment G31, wherein the sushi domain from human ILl5Ra comprises SEQ ID NO: 10.

Embodiment G33. The multi-chain chimeric polypeptide of embodiment G28, wherein the sushi domain from human ILl5Ra is a mature full-length ILl5Ra.

Embodiment G34. The multi-chain chimeric polypeptide of any one of embodiments G1-G21, wherein the pair of affinity domains is selected from the group consisting of: bamase and bamstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. Embodiment G35. The multi-chain chimeric polypeptide of any one of embodiments G1-G34, wherein the first target-binding domain and the second targeting binding domain each independently bind specifically to a receptor for IL-7, CD 16, or a receptor for IL-21. Embodiment G36. The multi-chain chimeric polypeptide of embodiment G35, wherein the first target-binding domain binds specifically to a receptor IL-7 and the second target-binding domain binds specifically to CD 16 or a receptor for IL-21. Embodiment G37. The multi-chain chimeric polypeptide of embodiment G36, wherein the first target-binding domain comprises a soluble IL-7 protein.

Embodiment G38. The multi-chain chimeric polypeptide of embodiment G37, wherein the soluble IL-7 protein is a soluble human IL-7.

Embodiment G39. The multi-chain chimeric polypeptide of any one of embodiments G36-G38, wherein the second antigen-binding domain comprises an antigen-binding domain that binds specifically to CD 16. Embodiment G40. The multi-chain chimeric polypeptide of embodiment G39, wherein the second antigen-binding domain comprises an scFv that binds specifically to CD 16.

Embodiment G41. The multi-chain chimeric polypeptide of any one of embodiments G36-G38, wherein the second antigen-binding domain bind specifically to a receptor for IL-21.

Embodiment G42. The multi-chain chimeric polypeptide of embodiment G41, wherein the second antigen-binding domain comprises a soluble IL-21.

Embodiment G43. The multi-chain chimeric polypeptide of embodiment G42, wherein the soluble IL-21 is a soluble human IL-21.

Embodiment G44. The multi-chain chimeric polypeptide of any one of embodiments G36-G40, wherein the second chimeric polypeptide further comprises an additional target-binding domain that binds specifically to a receptor for IL-21. Embodiment G45. The multi-chain chimeric polypeptide of embodiment G44, wherein the additional target-binding domain comprises a soluble IL-21.

Embodiment G46. The multi-chain chimeric polypeptide of embodiment G45, wherein the soluble IL-21 is a soluble human IL-12.

Embodiment G47. The multi-chain chimeric polypeptide of any one of embodiments G1-G34, wherein the first target-binding domain and the second targeting binding domain each independently bind specifically to TGF-b, CD16, or a receptor for IL-21.

Embodiment G48. The multi-chain chimeric polypeptide of embodiment G47, wherein the first target-binding domain binds specifically to a TGF-b and the second target-binding domain binds specifically to CD 16 or a receptor of IL-21.

Embodiment G49. The multi-specific chimeric polypeptide of embodiment G48, wherein the first target-binding domain is a soluble TGF-b receptor.

Embodiment G50. The multi-specific chimeric polypeptide of embodiment G49, wherein soluble TGF-b receptor is a soluble TGE^RII receptor.

Embodiment G51. The multi-specific chimeric polypeptide of any one of embodiments G48-G50, wherein the second target-binding domain binds specifically to CD 16.

Embodiment G52. The multi-specific chimeric polypeptide of embodiment G51, wherein the second antigen-binding domain comprises an antigen-binding domain that binds specifically to CD 16. Embodiment G53. The multi-chain chimeric polypeptide of embodiment G52, wherein the second antigen-binding domain comprises an scFv that binds specifically to CD 16. Embodiment G54. The multi-chain chimeric polypeptide of any one of embodiments G48-G50, wherein the second target-binding domain binds specifically to a receptor for IL-21.

Embodiment G55. The multi-chain chimeric polypeptide of embodiment G54, wherein the second target-binding domain comprises a soluble IL-21.

Embodiment G56. The multi-chain chimeric polypeptide of embodiment G55, wherein the second target-binding domain comprises a soluble human IL-21. Embodiment G57. The multi-chain chimeric polypeptide of any one of embodiments G48-G53, wherein the second chimeric polypeptide further comprises an additional target-binding domain that binds specifically to a receptor for IL-21.

Embodiment G58. The multi-chain chimeric polypeptide of embodiment G57, wherein the additional target-binding domain comprises a soluble IL-21.

Embodiment G59. The multi-chain chimeric polypeptide of embodiment G58, wherein the soluble IL-21 is a soluble human IL-21. Embodiment G60. The multi-chain chimeric polypeptide of any one of embodiments G1-G34, wherein the first target-binding domain and the second target- binding domain each independently bind specifically to a receptor for IL-7.

Embodiment G61. The multi-chain chimeric polypeptide of embodiment G60, wherein the first target-binding domain and the second target-binding domain include a soluble IL-7. Embodiment G62. The multi-chain chimeric polypeptide of embodiment G61, wherein the soluble IL-7 is a soluble human IL-7.

Embodiment G63. The multi-chain chimeric polypeptide of any one of embodiments G1-G34, wherein the first target-binding domain and the second target- binding domain each independently bind specifically to TGF-b.

Embodiment G64. The multi-specific chimeric polypeptide of embodiment G63, wherein the first target-binding domain and the second target-binding domain is a soluble TGF- b receptor.

Embodiment G65. The multi-specific chimeric polypeptide of embodiment G64, wherein the soluble TGF-b receptor is a soluble TGE^RII receptor. Embodiment G66. The multi-specific chimeric polypeptide of any one of embodiments G1-G34, wherein the first target-binding domain and the second targeting binding domain each independently bind specifically to a receptor for IL-7, a receptor for IL-21, or a receptor for CD137L. Embodiment G67. The multi-chain chimeric polypeptide of embodiment G66, wherein the first target-binding domain binds specifically to a receptor for IL-7 and the second target-binding domain binds specifically to a receptor for IL-21 or a receptor for CD137L. Embodiment G68. The multi-specific chimeric polypeptide of embodiment G67, wherein the first target-binding domain is a soluble IL-7.

Embodiment G69. The multi-specific chimeric polypeptide of embodiment G68, wherein the soluble IL-7 is a soluble human IL-7. Embodiment G70. The multi-chain chimeric polypeptide of any one of embodiments G67-G69, wherein the second target-binding domain binds specifically to a receptor for IL-21. Embodiment G71. The multi-chain chimeric polypeptide of embodiment G70, wherein the second target-binding domain is a soluble IL-21.

Embodiment G72. The multi-chain chimeric polypeptide of embodiment G71, wherein the soluble IL-21 is a soluble human IL-21.

Embodiment G73. The multi-chain chimeric polypeptide of any one of embodiments G67-G69, wherein the second antigen-binding domain binds specifically to a receptor for CD137L. Embodiment G74. The multi-chain chimeric polypeptide of embodiment G73, wherein the second antigen-binding domain is a soluble CD137L.

Embodiment G75. The multi-chain chimeric polypeptide of embodiment G74, wherein the soluble CD137L is a soluble human CD137L.

Embodiment G76. The multi-chain chimeric polypeptide of any one of embodiments G67-G72, wherein the second chimeric polypeptide further comprises an additional target-binding domain that binds specifically to a receptor for CD137L. Embodiment G77. The multi-chain chimeric polypeptide of embodiment G76, wherein the additional target-binding domain comprises a soluble CD137L.

Embodiment G78. The multi-chain chimeric polypeptide of embodiment G77, wherein the soluble CD137L is a soluble human CD137L. Embodiment G79. The multi-chain chimeric polypeptide of any one of embodiments G1-G34, wherein the first target-binding domain and the second targeting binding domain each independently bind specifically to a receptor for IL-7 or TGF-b. Embodiment G80. The multi-chain chimeric polypeptide of embodiment G79, wherein the first target-binding domain binds specifically to a receptor IL-7 and the second target-binding domain binds specifically to TGF-b.

Embodiment G81. The multi-chain chimeric polypeptide of embodiment G80, wherein the first target-binding domain comprises a soluble IL-7 protein.

Embodiment G82. The multi-chain chimeric polypeptide of embodiment G81, wherein the soluble IL-7 protein is a soluble human IL-7. Embodiment G83. The multi-chain chimeric polypeptide of any one of embodiments G80-G82, wherein the second antigen-binding domain comprises an antigen-binding domain that binds specifically to TGF-b.

Embodiment G84. The multi-specific chimeric polypeptide of embodiment G83, wherein the second target-binding domain is a soluble TGF-b receptor.

Embodiment G85. The multi-specific chimeric polypeptide of embodiment G84, wherein the soluble TGF-b receptor is a soluble TGE^RII receptor. Embodiment G86. The multi-chain chimeric polypeptide of any one of embodiments G1-G34, wherein the first target-binding domain and the second targeting binding domain each independently bind specifically to TGF-b, a receptor for IL-21, or a receptor for CD137L. Embodiment G87. The multi-chain chimeric polypeptide of embodiment G86, wherein the first target-binding domain binds specifically to a TGF-b and the second target-binding domain binds specifically to a receptor for IL-21 or a receptor for

CD137L.

Embodiment G88. The multi-specific chimeric polypeptide of embodiment G87, wherein the first target-binding domain is a soluble TGF-b receptor.

Embodiment G89. The multi-specific chimeric polypeptide of embodiment G88, wherein the soluble TGF-b receptor is a soluble TORbIIP receptor. Embodiment G90. The multi-specific chimeric polypeptide of any one of embodiments G87-G89, wherein the second target-binding domain binds specifically to a receptor for IL-21.

Embodiment G91. The multi-chain chimeric polypeptide of embodiment G90, wherein the second target-binding domain comprises a soluble IL-21.

Embodiment G92. The multi-chain chimeric polypeptide of embodiment G91, wherein the second target-binding domain comprises a soluble human IL-21. Embodiment G93. The multi-specific chimeric polypeptide of any one of embodiments G87-G89, wherein the second target-binding domain binds specifically to a receptor for CD137L.

Embodiment G94. The multi-chain chimeric polypeptide of embodiment G93, wherein the second target-binding domain comprises a soluble CD137L.

Embodiment G95. The multi-chain chimeric polypeptide of embodiment G94, wherein the second target-binding domain comprises a soluble human CD137L. Embodiment G96. The multi-chain chimeric polypeptide of any one of embodiments G87-G92, wherein the second chimeric polypeptide further comprises an additional target-binding domain that binds specifically to a receptor for CD137L. Embodiment G97. The multi-chain chimeric polypeptide of embodiment G96, wherein the additional target-binding domain comprises a soluble CD137L.

Embodiment G98. The multi-chain chimeric polypeptide of embodiment G97, wherein the soluble CD137L is a soluble human CD137L.

Embodiment G99. The multi-chain chimeric polypeptide of any one of embodiments G1-G34, wherein the first target-binding domain and the second targeting binding domain each independently bind specifically to TGF-b or a receptor for IL-21. Embodiment G100. The multi-chain chimeric polypeptide of embodiment G99, wherein the first target-binding domain binds specifically to a TGF-b and the second target-binding domain binds specifically to TGF-b or a receptor for IL-21.

Embodiment G101. The multi-specific chimeric polypeptide of embodiment G100, wherein the first target-binding domain is a soluble TGF-b receptor.

Embodiment G102. The multi-specific chimeric polypeptide of embodiment G101, wherein the soluble TGF-b receptor is a soluble TGE^RII receptor. Embodiment G103. The multi-specific chimeric polypeptide of any one of embodiments G100-G102, wherein the second target-binding domain binds specifically to a receptor for IL-21.

Embodiment G104. The multi-chain chimeric polypeptide of embodiment G103, wherein the second target-binding domain comprises a soluble IL-21. Embodiment G105. The multi-chain chimeric polypeptide of embodiment G104, wherein the second target-binding domain comprises a soluble human IL-21.

Embodiment G106. The multi-specific chimeric polypeptide of any one of embodiments G100-G102, wherein the second target-binding domain binds specifically to TGF-b.

Embodiment G107. The multi-specific chimeric polypeptide of embodiment G106, wherein the first target-binding domain is a soluble TGF-b receptor.

Embodiment G108. The multi-specific chimeric polypeptide of embodiment G107, wherein the soluble TGF-b receptor is a soluble TGE^RII receptor.

Embodiment G109. The multi-specific chimeric polypeptide of any one of embodiments G100-G105, wherein the second polypeptide further comprises an additional target-binding domain that binds specifically to TGF-b.

Embodiment G110. The multi-specific chimeric polypeptide of embodiment G109, wherein the first target-binding domain is a soluble TGF-b receptor.

Embodiment Gl 11. The multi-specific chimeric polypeptide of embodiment Gl 10, wherein the soluble TGF-b receptor is a soluble TGE^RII receptor.

Embodiment Gl 12. The multi-chain chimeric polypeptide of any one of embodiments G1-G34, wherein the first target-binding domain and the second targeting binding domain each independently bind specifically to TGF-b or IL-16.

Embodiment Gl 13. The multi-chain chimeric polypeptide of embodiment Gl 12, wherein the first target-binding domain binds specifically to a TGF-b and the second target-binding domain binds specifically to TGF-b or IL-16. Embodiment G114. The multi-specific chimeric polypeptide of embodiment Gl 13, wherein the first target-binding domain is a soluble TGF-b receptor.

Embodiment G115. The multi-specific chimeric polypeptide of embodiment Gl 14, wherein the soluble TGF-b receptor is a soluble TORbIIP receptor.

Embodiment Gl 16. The multi-specific chimeric polypeptide of any one of embodiments Gl 13-G115, wherein the second target-binding domain binds specifically to IL-16.

Embodiment G117. The multi-specific chimeric polypeptide of embodiment Gl 16, wherein the second antigen-binding domain comprises an antigen-binding domain that binds specifically to CD 16. Embodiment Gl 18. The multi-chain chimeric polypeptide of embodiment Gl 17, wherein the second antigen-binding domain comprises an scFv that binds specifically to CD 16.

Embodiment Gl 19. The multi-specific chimeric polypeptide of any one of embodiments Gl 13-G115, wherein the second target-binding domain binds specifically to TGF-b.

Embodiment G120. The multi-specific chimeric polypeptide of embodiment Gl 19, wherein the first target-binding domain is a soluble TGF-b receptor.

Embodiment G121. The multi-specific chimeric polypeptide of embodiment G120, wherein the soluble TGF-b receptor is a soluble TORbIIP receptor.

Embodiment G122. The multi-specific chimeric polypeptide of any one of embodiments Gl 13-G118, wherein the second chimeric polypeptide further comprises an additional target-binding domain that binds specifically to TGF-b. Embodiment G123. The multi-specific chimeric polypeptide of embodiment G122, wherein the first target-binding domain is a soluble TGF-b receptor.

Embodiment G124. The multi-specific chimeric polypeptide of embodiment G123, wherein the soluble TGF-b receptor is a soluble TORbIIP receptor.

Embodiment G125. The multi-chain chimeric polypeptide of any one of embodiments G1-G34, wherein the first target-binding domain and the second targeting binding domain each independently bind specifically to a TGF-b or a receptor for CD137L.

Embodiment G126. The multi-chain chimeric polypeptide of embodiment G125, wherein the first target-binding domain binds specifically to TGF-b and the second target-binding domain binds specifically to a receptor for CD137L.

Embodiment G127. The multi-specific chimeric polypeptide of embodiment G126, wherein the first target-binding domain is a soluble TGF-b receptor.

Embodiment G128. The multi-specific chimeric polypeptide of embodiment G127, wherein the soluble TGF-b receptor is a soluble TGE^RII receptor.

Embodiment G129. The multi-chain chimeric polypeptide of embodiment G128, wherein the second target-binding domain comprises a soluble CD137L protein. Embodiment G130. The multi-chain chimeric polypeptide of embodiment G129, wherein the soluble CD137L protein is a soluble human CD137L.

Embodiment G131. The multi-chain chimeric polypeptide of any one of embodiments G126-G130, wherein the second chimeric polypeptide further comprises an additional target-binding domain that binds specifically to TGF-b. Embodiment G132. The multi-specific chimeric polypeptide of embodiment G131, wherein the additional target-binding domain is a soluble TGF-b receptor.

Embodiment G133. The multi-specific chimeric polypeptide of embodiment G132, wherein the soluble TGF-b receptor is a soluble TORbIIP receptor.

Embodiment G134. The multi-chain chimeric polypeptide of embodiment Gl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 141.

Embodiment G135. The multi-chain chimeric polypeptide of embodiment G134, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 141. Embodiment G136. The multi-chain chimeric polypeptide of embodiment G135, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 141.

Embodiment G137. The multi-chain chimeric polypeptide of embodiment G136, wherein the first chimeric polypeptide comprises SEQ ID NO: 141.

Embodiment G138. The multi-chain chimeric polypeptide of embodiment G137, wherein the first chimeric polypeptide comprises SEQ ID NO: 143. Embodiment G139. The multi-chain chimeric polypeptide of any one of embodiments Gl and G134-G138, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 157.

Embodiment G140. The multi-chain chimeric polypeptide of embodiment G139, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 157. Embodiment G141. The multi-chain chimeric polypeptide of embodiment G140, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 157. Embodiment G142. The multi-chain chimeric polypeptide of embodiment G141, wherein the second chimeric polypeptide comprises SEQ ID NO: 157.

Embodiment G143. The multi-chain chimeric polypeptide of embodiment G142, wherein the second chimeric polypeptide comprises SEQ ID NO: 159.

Embodiment G144. The multi-chain chimeric polypeptide of embodiment Gl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 161. Embodiment G145. The multi-chain chimeric polypeptide of embodiment G144, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 161.

Embodiment G146. The multi-chain chimeric polypeptide of embodiment G145, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 161.

Embodiment G147. The multi-chain chimeric polypeptide of embodiment G146, wherein the first chimeric polypeptide comprises SEQ ID NO: 161.

Embodiment G148. The multi-chain chimeric polypeptide of embodiment G147, wherein the first chimeric polypeptide comprises SEQ ID NO: 163.

Embodiment G149. The multi-chain chimeric polypeptide of any one of embodiments Gl and G144-G148, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 157. Embodiment G150. The multi-chain chimeric polypeptide of embodiment G149, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 157. Embodiment Gl 51. The multi-chain chimeric polypeptide of embodiment Gl 50, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 157.

Embodiment G152. The multi-chain chimeric polypeptide of embodiment G151, wherein the second chimeric polypeptide comprises SEQ ID NO: 157.

Embodiment G153. The multi-chain chimeric polypeptide of embodiment G152, wherein the second chimeric polypeptide comprises SEQ ID NO: 159. Embodiment G154. The multi-chain chimeric polypeptide of embodiment Gl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 141.

Embodiment G155. The multi-chain chimeric polypeptide of embodiment G154, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 141.

Embodiment G156. The multi-chain chimeric polypeptide of embodiment G155, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 141.

Embodiment G157. The multi-chain chimeric polypeptide of embodiment G156, wherein the first chimeric polypeptide comprises SEQ ID NO: 141. Embodiment G158. The multi-chain chimeric polypeptide of embodiment G157, wherein the first chimeric polypeptide comprises SEQ ID NO: 143. Embodiment G159. The multi-chain chimeric polypeptide of any one of embodiments Gl and G154-G158, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 137. Embodiment G160. The multi-chain chimeric polypeptide of embodiment G159, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 137.

Embodiment G161. The multi-chain chimeric polypeptide of embodiment G160, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 137.

Embodiment G162. The multi-chain chimeric polypeptide of embodiment G161, wherein the second chimeric polypeptide comprises SEQ ID NO: 137.

Embodiment G163. The multi-chain chimeric polypeptide of embodiment G162, wherein the second chimeric polypeptide comprises SEQ ID NO: 139.

Embodiment G164. The multi-chain chimeric polypeptide of embodiment Gl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 161.

Embodiment G165. The multi-chain chimeric polypeptide of embodiment G164, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 161.

Embodiment G166. The multi-chain chimeric polypeptide of embodiment G165, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 161. Embodiment G167. The multi-chain chimeric polypeptide of embodiment G166, wherein the first chimeric polypeptide comprises SEQ ID NO: 161.

Embodiment G168. The multi-chain chimeric polypeptide of embodiment G167, wherein the first chimeric polypeptide comprises SEQ ID NO: 163.

Embodiment G169. The multi-chain chimeric polypeptide of any one of embodiments Gl and G164-G168, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 130.

Embodiment G170. The multi-chain chimeric polypeptide of embodiment G169, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 130. Embodiment G171. The multi-chain chimeric polypeptide of embodiment G170, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 130.

Embodiment G172. The multi-chain chimeric polypeptide of embodiment G171, wherein the second chimeric polypeptide comprises SEQ ID NO: 130.

Embodiment G173. The multi-chain chimeric polypeptide of embodiment G172, wherein the second chimeric polypeptide comprises SEQ ID NO: 132. Embodiment G174. The multi-chain chimeric polypeptide of embodiment Gl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 141.

Embodiment G175. The multi-chain chimeric polypeptide of embodiment G174, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 141. Embodiment G176. The multi-chain chimeric polypeptide of embodiment G175, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 141.

Embodiment G177. The multi-chain chimeric polypeptide of embodiment G176, wherein the first chimeric polypeptide comprises SEQ ID NO: 141.

Embodiment G178. The multi-chain chimeric polypeptide of embodiment G177, wherein the first chimeric polypeptide comprises SEQ ID NO: 143.

Embodiment G179. The multi-chain chimeric polypeptide of any one of embodiments Gl and G174-G178, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 169.

Embodiment G180. The multi-chain chimeric polypeptide of embodiment G179, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 169. Embodiment G181. The multi-chain chimeric polypeptide of embodiment G180, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 169.

Embodiment G182. The multi-chain chimeric polypeptide of embodiment G181, wherein the second chimeric polypeptide comprises SEQ ID NO: 169.

Embodiment G183. The multi-chain chimeric polypeptide of embodiment G182, wherein the second chimeric polypeptide comprises SEQ ID NO: 171. Embodiment G184. The multi-chain chimeric polypeptide of embodiment Gl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 141 Embodiment G185. The multi-chain chimeric polypeptide of embodiment G184, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 141.

Embodiment G186. The multi-chain chimeric polypeptide of embodiment G185, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 141.

Embodiment G187. The multi-chain chimeric polypeptide of embodiment G186, wherein the first chimeric polypeptide comprises SEQ ID NO: 141.

Embodiment G188. The multi-chain chimeric polypeptide of embodiment G187, wherein the first chimeric polypeptide comprises SEQ ID NO: 143.

Embodiment G189. The multi-chain chimeric polypeptide of any one of embodiments Gl and G184-G188, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 173.

Embodiment G190. The multi-chain chimeric polypeptide of embodiment G189, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 173.

Embodiment G191. The multi-chain chimeric polypeptide of embodiment G190, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 173. Embodiment G192. The multi-chain chimeric polypeptide of embodiment G191, wherein the second chimeric polypeptide comprises SEQ ID NO: 173.

Embodiment G193. The multi-chain chimeric polypeptide of embodiment G192, wherein the second chimeric polypeptide comprises SEQ ID NO: 175.

Embodiment G194. The multi-chain chimeric polypeptide of embodiment Gl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 141.

Embodiment G195. The multi-chain chimeric polypeptide of embodiment G194, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 141. Embodiment G196. The multi-chain chimeric polypeptide of embodiment G195, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 141.

Embodiment G197. The multi-chain chimeric polypeptide of embodiment G196, wherein the first chimeric polypeptide comprises SEQ ID NO: 141.

Embodiment G198. The multi-chain chimeric polypeptide of embodiment G197, wherein the first chimeric polypeptide comprises SEQ ID NO: 143. Embodiment G199. The multi-chain chimeric polypeptide of any one of embodiments Gl and G194-G198, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 129.

Embodiment G200. The multi-chain chimeric polypeptide of embodiment G199, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 130. Embodiment G201. The multi-chain chimeric polypeptide of embodiment G200, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 130. Embodiment G202. The multi-chain chimeric polypeptide of embodiment G201, wherein the second chimeric polypeptide comprises SEQ ID NO: 130.

Embodiment G203. The multi-chain chimeric polypeptide of embodiment G202, wherein the second chimeric polypeptide comprises SEQ ID NO: 132.

Embodiment G204. The multi-chain chimeric polypeptide of embodiment Gl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 161. Embodiment G205. The multi-chain chimeric polypeptide of embodiment G204, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 161.

Embodiment G206. The multi-chain chimeric polypeptide of embodiment G205, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 161.

Embodiment G207. The multi-chain chimeric polypeptide of embodiment G206, wherein the first chimeric polypeptide comprises SEQ ID NO: 161.

Embodiment G208. The multi-chain chimeric polypeptide of embodiment G207, wherein the first chimeric polypeptide comprises SEQ ID NO: 163.

Embodiment G209. The multi-chain chimeric polypeptide of any one of embodiments Gl and G204-G208, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 169. Embodiment G210. The multi-chain chimeric polypeptide of embodiment G209, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 169. Embodiment G211. The multi-chain chimeric polypeptide of embodiment G210, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 169.

Embodiment G212. The multi-chain chimeric polypeptide of embodiment G211, wherein the second chimeric polypeptide comprises SEQ ID NO: 169.

Embodiment G213. The multi-chain chimeric polypeptide of embodiment G212, wherein the second chimeric polypeptide comprises SEQ ID NO: 171. Embodiment G214. The multi-chain chimeric polypeptide of embodiment Gl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 161.

Embodiment G215. The multi-chain chimeric polypeptide of embodiment G214, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 161.

Embodiment G216. The multi-chain chimeric polypeptide of embodiment G215, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 161.

Embodiment G217. The multi-chain chimeric polypeptide of embodiment GE216, wherein the first chimeric polypeptide comprises SEQ ID NO: 161. Embodiment G218. The multi-chain chimeric polypeptide of embodiment G217, wherein the first chimeric polypeptide comprises SEQ ID NO: 163. Embodiment G219. The multi-chain chimeric polypeptide of any one of embodiments Gl and G214-G218, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 177. Embodiment G220. The multi-chain chimeric polypeptide of embodiment G219, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 177.

Embodiment G221. The multi-chain chimeric polypeptide of embodiment G220, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 177.

Embodiment G222. The multi-chain chimeric polypeptide of embodiment G221, wherein the second chimeric polypeptide comprises SEQ ID NO: 177.

Embodiment G223. The multi-chain chimeric polypeptide of embodiment G222, wherein the second chimeric polypeptide comprises SEQ ID NO: 179.

Embodiment G224. The multi-chain chimeric polypeptide of embodiment Gl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 161.

Embodiment G225. The multi-chain chimeric polypeptide of embodiment G224, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 161.

Embodiment G226. The multi-chain chimeric polypeptide of embodiment G225, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 161. Embodiment G227. The multi-chain chimeric polypeptide of embodiment G226, wherein the first chimeric polypeptide comprises SEQ ID NO: 161.

Embodiment G228. The multi-chain chimeric polypeptide of embodiment G227, wherein the first chimeric polypeptide comprises SEQ ID NO: 163.

Embodiment G229. The multi-chain chimeric polypeptide of any one of embodiments Gl and G224-G228, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 181.

Embodiment G230. The multi-chain chimeric polypeptide of embodiment G229, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 181. Embodiment G231. The multi-chain chimeric polypeptide of embodiment G230, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 181.

Embodiment G232. The multi-chain chimeric polypeptide of embodiment G231, wherein the second chimeric polypeptide comprises SEQ ID NO: 181.

Embodiment G233. The multi-chain chimeric polypeptide of embodiment G232, wherein the second chimeric polypeptide comprises SEQ ID NO: 183. Embodiment G234. The multi-chain chimeric polypeptide of embodiment Gl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 161.

Embodiment G235. The multi-chain chimeric polypeptide of embodiment G234, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 161. Embodiment G236. The multi-chain chimeric polypeptide of embodiment G235, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 161. Embodiment G237. The multi-chain chimeric polypeptide of embodiment G236, wherein the first chimeric polypeptide comprises SEQ ID NO: 161.

Embodiment G238. The multi-chain chimeric polypeptide of embodiment G237, wherein the first chimeric polypeptide comprises SEQ ID NO: 163.

Embodiment G239. The multi-chain chimeric polypeptide of any one of embodiments Gl and G234-G238, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 185 Embodiment G240. The multi-chain chimeric polypeptide of embodiment G239, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 185.

Embodiment G241. The multi-chain chimeric polypeptide of embodiment G240, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 185.

Embodiment G242. The multi-chain chimeric polypeptide of embodiment G241, wherein the second chimeric polypeptide comprises SEQ ID NO: 185.

Embodiment G243. The multi-chain chimeric polypeptide of embodiment G242, wherein the second chimeric polypeptide comprises SEQ ID NO: 187.

Embodiment G244. The multi-chain chimeric polypeptide of any one of embodiments G1-G133, wherein the first chimeric polypeptide further comprises one or more additional target-binding domain(s), where at least one of the one or more additional antigen-binding domain(s) is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment G245. The multi-chain chimeric polypeptide of embodiment G244, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the at least one of the one or more additional antigen binding domain(s), and/or a linker sequence between the at least one of the one or more additional antigen-binding domain(s)and the first domain of the pair of affinity domains. Embodiment G246. The multi-chain chimeric polypeptide of any one of embodiments G1-G133, wherein the first chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide. Embodiment G247. The multi-chain chimeric polypeptide of embodiment G246, wherein at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment G248. The multi-chain chimeric polypeptide of embodiment G246, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains.

Embodiment G249. The multi-chain chimeric polypeptide of embodiment G246, wherein the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment G250. The multi-chain chimeric polypeptide of embodiment G246, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first target-binding domain. Embodiment G251. The multi-chain chimeric polypeptide of embodiment G246, wherein at least one of the one or more additional target-binding domains is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment G252. The multi-chain chimeric polypeptide of embodiment G251, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment G253. The multi-chain chimeric polypeptide of embodiment G251, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment G254. The multi-chain chimeric polypeptide of embodiment G251, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment G255. The multi-chain chimeric polypeptide of embodiment G251, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment G256. The multi-chain chimeric polypeptide of embodiment G251, wherein the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, directly abuts the soluble tissue factor domain and/or the first domain of the pair of affinity domains.

Embodiment G257. The multi-chain chimeric polypeptide of embodiment G251, wherein the first chimeric polypeptide further comprises a linker sequence disposed (i) between the soluble tissue factor domain and the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target- binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment G258. The multi-chain chimeric polypeptide of any one of embodiments G44-G46, G57-G59, G76-G78, G96-G98, G109-G111, G122-G124, and G131-G133, wherein the second chimeric polypeptide further comprises the additional target-binding domain at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment G259. The multi-chain chimeric polypeptide of embodiment G258, wherein the additional target-binding domain directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment G260. The multi-chain chimeric polypeptide of embodiment G258, wherein the second chimeric polypeptide further comprises a linker sequence between the additional target-binding domain and the second domain of the pair of affinity domains in the second chimeric polypeptide. Embodiment G261. The multi-chain chimeric polypeptide of embodiment G258, wherein the additional target-binding domain directly abuts the second target-binding domain in the second chimeric polypeptide. Embodiment G262. The multi-chain chimeric polypeptide of embodiment G258, wherein the second chimeric polypeptide further comprises a linker sequence between the additional target-binding domain and the second target-binding domain in the second chimeric polypeptide. Embodiment G263. A composition comprising any of the multi-chain chimeric polypeptides of embodiments G1-G262.

Embodiment G264. The composition of embodiment G263, wherein the composition is a pharmaceutical composition.

Embodiment G265. A kit comprising at least one dose of the composition of embodiment G263 or G264.

Embodiment G266. Nucleic acid encoding any of the multi-chain chimeric polypeptides of any one of embodiments G1-G262.

Embodiment G267. A vector comprising the nucleic acid of embodiment G266.

Embodiment G268. The vector of embodiment G267, wherein the vector is an expression vector.

Embodiment G269. A cell comprising the nucleic acid of embodiment G266 or the vector of embodiment G267 or G268. Embodiment G270. A method of producing a multi-chain chimeric polypeptide, the method comprising: culturing the cell of embodiment G269 in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cell and/or the culture medium.

Embodiment G271. A multi-chain chimeric polypeptide produced by the method of embodiment G327.

Embodiment G272. The multi-chain chimeric polypeptide of embodiment G8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 5.

Embodiment G273. The multi-chain chimeric polypeptide of embodiment G272, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5.

Embodiment G274. The multi-chain chimeric polypeptide of embodiment G273, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 5.

Embodiment G275. The multi-chain chimeric polypeptide of embodiment G274, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 5.

Embodiment G276. The multi-chain chimeric polypeptide of embodiment G8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 6.

Embodiment G277. The multi-chain chimeric polypeptide of embodiment G276, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 6. Embodiment G278. The multi-chain chimeric polypeptide of embodiment G277, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 6.

Embodiment G279. The multi-chain chimeric polypeptide of embodiment G279, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 6.

Embodiment Hl. A method of promoting the activation and proliferation of a natural killer cell or a T cell, the method comprising:

contacting a natural killer cell or a T cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target- binding domain, a linker domain, and a second target-binding domain, and (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for the activation and proliferation of the natural killer cell or the T cell,

wherein the first target-binding domain and the second target-binding domain are each independently selected from the group consisting of: a soluble interleukin or cytokine protein, an antigen-binding domain, a soluble interleukin or cytokine receptor, and ligands of co-stimulatory molecules.

Embodiment H2. The method of embodiment Hl, wherein the first target-binding domain and the linker domain directly abut each other.

Embodiment H3. The method of embodiment Hl, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the linker domain.

Embodiment H4. The method of any one of embodiments H1-H3, wherein the linker domain and the second target-binding domain directly abut each other.

Embodiment H5. The method of any one of embodiments H1-H3, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the linker domain and the second target-binding domain.

Embodiment H6. The method of embodiment Hl, wherein the first target-binding domain and the second target-binding domain directly abut each other. Embodiment H7. The method of embodiment Hl, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the second target-binding domain. Embodiment H8. The method of embodiment H6 or H7, wherein the second target-binding domain and the linker domain directly abut each other.

Embodiment H9. The method of embodiment H6 or H7, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the second target- binding domain and the linker domain.

Embodiment H10. The method of any one of embodiments H1-H9, wherein the first target-binding domain and the second target-binding domain bind specifically to the same antigen.

Embodiment Hl 1. The method of embodiment H10, wherein the first target- binding domain and the second target-binding domain bind specifically to the same epitope. Embodiment H12. The method of embodiment Hl 1, wherein the first target- binding domain and the second target-binding domain comprise the same amino acid sequence.

Embodiment H13. The method of any one of embodiments H1-H9, wherein the first target-binding domain and the second target-binding domain bind specifically to different antigens.

Embodiment H14. The method of any one of embodiments H1-H13, wherein one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain. Embodiment H15. The method of embodiment H14, wherein the first target- binding domain and the second target-binding domain are each an antigen-binding domain. Embodiment H16. The method of embodiment H14 or H15, wherein antigen binding domain comprises a scFv or a single domain antibody.

Embodiment H17. The method of any one of embodiments H1-H16, wherein one or both of the first target-binding domain and the second target-binding domain bind to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD52, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-pRII), a ligand of TGF-pRIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-l 5, a receptor for IL- 17, a receptor for IL-l 8, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.

Embodiment H18. The method of any one of embodiments H1-H16, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. Embodiment H19. The method of embodiment H18, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL- 21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULPl6-binding protein.

Embodiment H20. The method of any one of embodiments H1-H16, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor.

Embodiment H21. The method of embodiment H20, wherein the soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor is a soluble TGF-b receptor II (TGF-PRII), a soluble TGF-PRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28.

Embodiment H22. The method of any one of embodiments H1-H21, wherein the linker domain is a soluble tissue factor domain.

Embodiment H23. The method of embodiment H22, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment H24. The method of embodiment H23, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1.

Embodiment H25. The method of embodiment H24, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment H26. The method of embodiment H25, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1. Embodiment H27. The method of any one of embodiments H23-H26, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment H28. The method of embodiment H27, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment H29. The method of any one of embodiments H22-H28, wherein the soluble tissue factor domain is not capable of binding Factor Vila.

Embodiment H30. The method of any one of embodiments H22-H29, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. Embodiment H31. The method of any one of embodiments H22-H30, wherein the single-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

Embodiment H32. The method of any one of embodiments H22-H31, wherein the IgGl antibody construct comprises at least one antigen-binding domain that binds specifically to the soluble tissue factor domain.

Embodiment H33. The method of any one of embodiments H1-H21, wherein the linker domain is selected from the group consisting of: a kappa chain and a lambda chain. Embodiment H34. The method of any one of embodiments H1-H33, wherein the

IgGl antibody construct is a monoclonal IgGl antibody, where both of the antigen binding domains in the monoclonal IgGl antibody bind specifically to the linker domain.

Embodiment H35. The method of any one of embodiments H1-H33, wherein the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen binding domains in the bispecific IgGl antibody binds specifically to the linker domain.

Embodiment H36. The method of any one of embodiments H1-H35, wherein the single-chain chimeric polypeptide further comprises one or more additional target- binding domains at its N- and/or C-terminus. Embodiment H37. The method of embodiment H36, wherein the single-chain chimeric polypeptide comprises one or more additional target-binding domains at its N- terminus. Embodiment H38. The method of embodiment H37, wherein one or more additional target-binding domains directly abuts the first target-binding domain, the second target-binding domain, or the linker domain.

Embodiment H39. The method of embodiment H38, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target-binding domains and the first target-binding domain, the second target- binding domain, or the linker domain.

Embodiment H40. The method of embodiment H36, wherein the single-chain chimeric polypeptide comprises one or more additional target-binding domains at its C- terminus.

Embodiment H41. The method of embodiment H40, wherein one of the one or more additional target-binding domains directly abuts the first target-binding domain, the second target-binding domain, or the linker domain.

Embodiment H42. The method of embodiment H40, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target-binding domains and the first target-binding domain, the second target- binding domain, or the linker domain.

Embodiment H43. The method of embodiment H36, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N- terminus and the C-terminus. Embodiment H44. The method of embodiment H43, wherein one of the one or more additional target-binding domains at the N-terminus directly abuts the first target- binding domain, the second target-binding domain, or the linker domain. Embodiment H45. The method of embodiment H43, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional target-binding domains at the N-terminus and the first target-binding domain, the second target-binding domain, or the linker domain. Embodiment H46. The method of embodiment H43, wherein one of the one or more additional target-binding domains at the C-terminus directly abuts the first target- binding domain, the second target-binding domain, or the linker domain.

Embodiment H47. The method of embodiment H43, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional target-binding domains at the C-terminus and the first target-binding domain, the second target-binding domain, or the linker domain.

Embodiment H48. The method of any one of embodiments H36-H47, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen.

Embodiment H49. The method of embodiment H48, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope.

Embodiment H50. The method of embodiment H49, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence. Embodiment H51. The method of embodiment H48, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains each bind specifically to the same antigen.

Embodiment H52. The method of embodiment H51, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains each bind specifically to the same epitope.

Embodiment H53. The method of embodiment H52, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains each comprise the same amino acid sequence.

Embodiment H54. The method of any one of embodiments H36-H47, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to different antigens.

Embodiment H55. The method of any one of embodiments H36-H54, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen-binding domain.

Embodiment H56. The method of embodiment H55, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains are each an antigen-binding domain.

Embodiment H57. The method of embodiment H55 or H56, wherein the antigen binding domain comprises a scFv or a single domain antibody.

Embodiment H58. The method of any one of embodiments H36-H57, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD 137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-PRII), a ligand of TGF-PRIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD28.

Embodiment H59. The method of any one of embodiments H36-H57, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein.

Embodiment H60. The method of embodiment H59, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL- 21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment H61. The method of any one of embodiments H36-H57, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor. Embodiment H62. The method of embodiment H61, wherein the soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface receptor is a soluble TGF-b receptor II (TGF-PRII), a soluble TGF-PRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28.

Embodiment H63. The method of any one of embodiments H36-H57, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a ligand of a co-stimulatory molecule.

Embodiment H64. The method of embodiment H63, wherein the ligand of a co- stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l.

Embodiment H65. The method of any one of embodiments H1-H64, wherein the single-chain chimeric polypeptide further comprises a peptide tag positioned at the N- terminal end or the C-terminal end of the single-chain chimeric polypeptide.

Embodiment H66. The method of any one of embodiments H1-H65, wherein the contacting step is performed for a period of about 2 hours to about 20 days.

Embodiment H67. The method of embodiment H66, wherein the contacting step is performed for a period of about 1 day to about 15 days.

Embodiment H68. The method of any one of embodiments H1-H67, wherein the liquid culture medium is a serum-free liquid culture medium.

Embodiment H69. The method of any one of embodiments H1-H67, wherein the liquid culture medium is a chemically-defined liquid culture medium. Embodiment H70. The method of any one of embodiments H1-H69, wherein the liquid culture medium comprises the single-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0.5: 1 to about 2: 1. Embodiment H71. The method of embodiment H70, wherein the liquid culture medium comprises the single-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0.8:1 to about 1.2: 1.

Embodiment H72. The method of any one of embodiments H1-H71, wherein the NK cell or T cell was previously obtained from a subject.

Embodiment H73. The method of embodiment H72, wherein the method further comprises obtaining the NK cell or T cell from the subject prior to the contacting step. Embodiment H74. The method of any one of embodiments H1-H73, wherein the

NK cell or T cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

Embodiment H75. The method of any one of embodiments H1-H73, wherein the method further comprises, after the contacting step, introducing into the NK cell or the T cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Embodiment H76. The method of any one of embodiments H1-H73, wherein the method further comprises, before the contacting step, introducing into the NK cell or the T cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Embodiment H77. The method of any one of embodiments H1-H76, wherein the method further comprises, after the contacting step, isolating the NK cell or the T cell. Embodiment H78. The method of any one of embodiments H1-H77, wherein after the contacting step, the NK cell or the T cell has an increased level of expression or secretion of one or more proteins selected from the group consisting of: TNF-a, IFN-T, granzyme A, granzyme B, perforin, 2B4, CD8, CDl la, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, CDl58a, CDl58b, CDl58e, CD178, CD226, CD253, NKG2A, NKG2C, NKG2D, LIR-l, LILR-B1, KIR2DL1, KIR3DL1, KIR2DL2, KIR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-l, NKG2A, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-a, and IFN-g as compared to the level of expression or secretion of the one or more proteins prior to the contacting step.

Embodiment H79. The method of any one of embodiments H1-H78, wherein the method further comprises, after the contacting step, administering the NK cell or the T cell to a subject in need thereof.

Embodiment H80. The method of embodiment H79, wherein the subject has been identified or diagnosed as having an age-related disease or condition.

Embodiment H81. The method of embodiment H80, wherein the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. Embodiment H82. The method of embodiment H79, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment H83. The method of embodiment H82, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment H84. The method of embodiment H79, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment H85. The method of embodiment H84, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment H86. An activated NK cell or T cell produced by the method of any one of embodiments H1-H78.

Embodiment H87. A pharmaceutical composition comprising the activated NK cell or the activated T cell of embodiment H86. Embodiment H88. A kit comprising a pharmaceutical composition comprising the activated NK cell or the activated T cell of embodiment H86.

Embodiment H89. A method of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the activated NK cell or the activated T cell of embodiment H86 or the pharmaceutical composition of embodiment H87.

Embodiment H90. The method of embodiment H89, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment H91. The method of embodiment H90, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment H92. The method of embodiment H89, wherein the subject has been identified or diagnosed as having an aging-related disease or condition.

Embodiment H93. The method of embodiment H92, wherein the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment H94. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the activated NK cell or the activated T cell of embodiment H86 or the pharmaceutical composition of embodiment H87.

Embodiment H95. The method of embodiment H94, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment H96. The method of embodiment H95, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment H97. The method of embodiment H94, wherein the subject has been identified or diagnosed as having an aging-related disease or condition. Embodiment H98. The method of embodiment H97, wherein the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment H99. The method of embodiment H94, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment H100. The method of embodiment H99, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment H101. A kit comprising:

(i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, wherein the first target-binding domain and the second target-binding domain are each independently selected from the group consisting of: a soluble interleukin or cytokine protein, an antigen-binding domain, a soluble interleukin or cytokine receptor, and ligands of co-stimulatory molecules; and

(ii) an IgGl antibody construct comprising at least one antigen-binding domain that binds specifically to the linker domain. Embodiment H102. The kit of embodiment H101, wherein the first target-binding domain and the linker domain directly abut each other. Embodiment H103. The kit of embodiment H101, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the linker domain. Embodiment H104. The kit of any one of embodiments H101-H103, wherein the linker domain and the second target-binding domain directly abut each other.

Embodiment H105. The kit of any one of embodiments H101-H103, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the linker domain and the second target-binding domain.

Embodiment H106. The kit of embodiment H101, wherein the first target-binding domain and the second target-binding domain directly abut each other. Embodiment H107. The kit of embodiment H101, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the second target-binding domain.

Embodiment H108. The kit of embodiment H106 or H107, wherein the second target-binding domain and the linker domain directly abut each other.

Embodiment H109. The kit of embodiment H106 or H107, wherein the single- chain chimeric polypeptide further comprises a linker sequence between the second target-binding domain and the linker domain.

Embodiment Hl 10. The kit of any one of embodiments H101-H109, wherein the first target-binding domain and the second target-binding domain bind specifically to the same antigen. Embodiment Hl 11. The kit of embodiment Hl 10, wherein the first target-binding domain and the second target-binding domain bind specifically to the same epitope. Embodiment Hl 12. The kit of embodiment Hl 11, wherein the first target-binding domain and the second target-binding domain comprise the same amino acid sequence.

Embodiment Hl 13. The kit of any one of embodiments H101-H109, wherein the first target-binding domain and the second target-binding domain bind specifically to different antigens.

Embodiment Hl 14. The kit of any one of embodiments H101-H113, wherein one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain.

Embodiment Hl 15. The kit of embodiment Hl 14, wherein the first target-binding domain and the second target-binding domain are each an antigen-binding domain. Embodiment Hl 16. The kit of embodiment Hl 14 or Hl 15, wherein the antigen binding domain comprises a scFv or a single domain antibody.

Embodiment Hl 17. The kit of any one of embodiments H101-H116, wherein one or both of the first target-binding domain and the second target-binding domain bind to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin,

CEACAM5, a ULl6-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-pRII), a ligand of TGF-pRIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-l 5, a receptor for IL- 17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.

Embodiment Hl 18. The kit of any one of embodiments H101-H116, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. Embodiment Hl 19. The kit of embodiment Hl 18, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein. Embodiment H120. The kit of any one of embodiments H101-H116, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin or cytokine receptor.

Embodiment H121. The kit of embodiment H120, wherein the soluble interleukin or cytokine receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble

DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28.

Embodiment H122. The kit of any one of embodiments H101-H121, wherein the linker domain is a soluble tissue factor domain.

Embodiment H123. The kit of embodiment H122, wherein the soluble tissue factor domain is a soluble human tissue factor domain. Embodiment H124. The kit of embodiment H123, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1. Embodiment H125. The kit of embodiment H124, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment H126. The kit of embodiment H125, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1.

Embodiment H127. The kit of any one of embodiments H123-H126, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment H128. The kit of embodiment H127, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. Embodiment H129. The kit of any one of embodiments H122-H128, wherein the soluble tissue factor domain is not capable of binding Factor Vila.

Embodiment H130. The kit of any one of embodiments H122-H129, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment H131. The kit of any one of embodiments H122-H130, wherein the single-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

Embodiment H132. The kit of any one of embodiments H122-H131, wherein the IgGl antibody construct comprises at least one antigen-binding domain that binds specifically to the soluble tissue factor domain.

Embodiment H133. The kit of any one of embodiments H101-H121, wherein the linker domain is selected from the group consisting of: a kappa chain and a lambda chain.

Embodiment H134. The kit of any one of embodiments H101-H133, wherein the IgGl antibody construct is a monoclonal IgGl antibody, where both antigen-binding domains in the monoclonal IgGl antibody bind specifically to the linker domain. Embodiment H135. The kit of any one of embodiments H101-H133, wherein the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen binding domains in the bispecific IgGl antibody binds specifically to the linker domain. Embodiment H136. The kit of any one of embodiments H101-H135, wherein the single-chain chimeric polypeptide further comprises one or more additional target- binding domains at its N- and/or C-terminus.

Embodiment H137. The kit of embodiment H136, wherein the single-chain chimeric polypeptide comprises one or more additional target-binding domains at its N- terminus.

Embodiment H138. The kit of embodiment H137, wherein one or more additional target-binding domains directly abuts the first target-binding domain, the second target- binding domain, or the linker domain.

Embodiment H139. The kit of embodiment H138, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target-binding domains and the first target-binding domain, the second target- binding domain, or the linker domain.

Embodiment H140. The kit of embodiment H136, wherein the single-chain chimeric polypeptide comprises one or more additional target-binding domains at its C- terminus.

Embodiment H141. The kit of embodiment H140, wherein one of the one or more additional target-binding domains directly abuts the first target-binding domain, the second target-binding domain, or the linker domain. Embodiment H142. The kit of embodiment H140, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target-binding domains and the first target-binding domain, the second target- binding domain, or the linker domain.

Embodiment H143. The kit of embodiment H136, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N- terminus and the C-terminus.

Embodiment H144. The kit of embodiment H143, wherein one of the one or more additional target-binding domains at the N-terminus directly abuts the first target-binding domain, the second target-binding domain, or the linker domain.

Embodiment H145. The kit of embodiment H143, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional target-binding domains at the N-terminus and the first target-binding domain, the second target-binding domain, or the linker domain.

Embodiment H146. The kit of embodiment H143, wherein one of the one or more additional target-binding domains at the C-terminus directly abuts the first target-binding domain, the second target-binding domain, or the linker domain.

Embodiment H147. The kit of embodiment H143, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional target-binding domains at the C-terminus and the first target-binding domain, the second target-binding domain, or the linker domain.

Embodiment H148. The kit of any one of embodiments H136-H147, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. Embodiment H149. The kit of embodiment H148, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope. Embodiment H150. The kit of embodiment H149, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

Embodiment H151. The kit of embodiment H148, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same antigen.

Embodiment H152. The kit of embodiment Ell 51, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same epitope.

Embodiment H153. The kit of embodiment H152, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each comprise the same amino acid sequence.

Embodiment H154. The kit of any one of embodiments H136-H147, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to different antigens. Embodiment H155. The kit of any one of embodiments H136-H154, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen-binding domain.

Embodiment H156. The kit of embodiment H155, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen-binding domain. Embodiment H157. The kit of embodiment H155 or H156, wherein the antigen binding domain comprises a scFv or a single domain antibody.

Embodiment H158. The kit of any one of embodiments H136-H157, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P- cadherin, CEACAM5, a EIL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD 122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-pRII), a ligand of TGF- PRIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-l 5, a receptor for IL- 17, a receptor for IL-l 8, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.

Embodiment H159. The kit of any one of embodiments H136-H157, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein.

Embodiment H160. The kit of embodiment H159, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP 16-binding protein. Embodiment H161. The kit of any one of embodiments H136-H157, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin or cytokine receptor.

Embodiment H162. The kit of embodiment H161, wherein the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28.

Embodiment H163. The method of any one of embodiments H136-H162, wherein one or both of the first target-binding domain and the second target-binding domain is a ligand of a co-stimulatory molecule.

Embodiment H164. The kit of embodiment H163, wherein the ligand of a co- stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l.

Embodiment H165. The kit of any one of embodiments H101-H164, wherein the single-chain chimeric polypeptide further comprises a peptide tag positioned at the N- terminal end or the C-terminal end of the single-chain chimeric polypeptide.

Embodiment H166. A method of promoting the activation and proliferation of a natural killer cell or a T cell, the method comprising:

contacting a natural killer cell or a T cell in a liquid culture medium comprising:

(1) an effective amount of a multi-chain chimeric polypeptide comprising:

(c) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a linker domain; and

(iii) a first domain of a pair of affinity domains;

(d) a second chimeric polypeptide comprising: (1) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and

(2) an effective amount of an IgGl antibody construct comprising at least one antigen-binding domain that binds specifically to the linker domain,

under conditions that allow for the activation and proliferation of the natural killer cell or the T cell.

Embodiment H167. The method of embodiment H166, wherein the first target- binding domain and the linker domain directly abut each other in the first chimeric polypeptide. Embodiment H168. The method of embodiment H166, wherein the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the linker domain in the first chimeric polypeptide.

Embodiment H169. The method of any one of embodiments Ell 66-Ell 68, wherein the linker domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide.

Embodiment Ell 70. The method of any one of embodiments Ell 66-Ell 68, wherein the first chimeric polypeptide further comprises a linker sequence between the linker domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment ET171. The method of any one of embodiments E1166-E1170, wherein the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. Embodiment H172. The method of any one of embodiments H166-H170, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

Embodiment H173. The method of any one of embodiments Ell 66-Ell 72, wherein the first target-binding domain and the second target-binding domain bind specifically to the same antigen. Embodiment E1174. The method of embodiment ET173, wherein the first target- binding domain and the second target-binding domain bind specifically to the same epitope.

Embodiment ET175. The method of embodiment E1174, wherein the first target- binding domain and the second target-binding domain comprise the same amino acid sequence.

Embodiment Ell 76. The method of any one of embodiments Ell 66-Ell 72, wherein the first target-binding domain and the second target-binding domain bind specifically to different antigens.

Embodiment Ell 77. The method of any one of embodiments Ell 66-Ell 76, wherein one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain.

Embodiment ET178. The method of embodiment E1177, wherein the first target binding domain and the second target-binding domain are each antigen-binding domains.

Embodiment Ell 79. The method of embodiment Ell 77 or Ell 78, wherein the antigen-binding domain comprises a scFv or a single domain antibody. Embodiment H180. The method of any one of embodiments H166-H179, wherein one or both of the first target-binding domain and the second target-binding domain bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD 19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P- cadherin, CEACAM5, a EIL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD 122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF- b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-l 5, a receptor for IL- 17, a receptor for IL-l 8, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28. Embodiment H181. The method of any one of embodiments H166-H179, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein.

Embodiment H182. The method of embodiment H181, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL- 21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment H183. The method of any one of embodiments H166-H179, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin or cytokine receptor. Embodiment H184. The method of embodiment H183, wherein the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF^RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28.

Embodiment H185. The method of any one of embodiments Ell 66-Ell 84, wherein the first chimeric polypeptide further comprises one or more additional target-binding domain(s), where at least one of the one or more target-binding domain(s) is positioned between the linker domain and the first domain of the pair of affinity domains.

Embodiment H186. The method of embodiment H185, wherein the first chimeric polypeptide further comprises a linker sequence between the linker domain and the at least one of the one or more target antigen-binding domain(s), and/or a linker sequence between the at least one of the one or more target antigen-binding domain(s) and the first domain of the pair of affinity domains.

Embodiment H187. The method of any one of embodiments Ell 66-Ell 84, wherein the first chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide.

Embodiment H188. The method of embodiment H187, wherein at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment H189. The method of embodiment H187, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains. Embodiment H190. The method of embodiment H187, wherein the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide. Embodiment H191. The method of embodiment H187, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first target-binding domain.

Embodiment H192. The method of embodiment H187, wherein at least one of the one or more additional target-binding domains is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target- binding domains is positioned between the linker domain and the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment H193. The method of embodiment H192, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment H194. The method of embodiment H192, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment H195. The method of embodiment H192, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment H196. The method of embodiment H192, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H197. The method of embodiment H192, wherein the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains, directly abuts the linker domain and/or the first domain of the pair of affinity domains.

Embodiment H198. The method of embodiment H192, wherein the first chimeric polypeptide further comprises a linker sequence disposed (i) between the linker domain and the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains.

Embodiment H199. The method of any one of embodiments Ell 66-Ell 98, wherein the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end and/or the C-terminal end of the second chimeric polypeptide.

Embodiment E1200. The method of embodiment Ell 99, wherein at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment E1201. The method of embodiment E1199, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide. Embodiment H202. The method of embodiment H199, wherein at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide. Embodiment H203. The method of embodiment H199, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second target-binding domain in the second chimeric polypeptide. Embodiment H204. The method of any one of embodiments H185-H203, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen.

Embodiment H205. The method of embodiment H204, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope.

Embodiment H206. The method of embodiment H205, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

Embodiment H207. The method of embodiment H204, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains each bind specifically to the same antigen.

Embodiment H208. The method of embodiment H207, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains each bind specifically to the same epitope. Embodiment H209. The method of embodiment H208, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains each comprise the same amino acid sequence.

Embodiment H210. The method of any one of embodiments H185-H203, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to different antigens.

Embodiment H211. The method of any one of embodiments H185-H210, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen-binding domain.

Embodiment H212. The method of embodiment H211, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains are each an antigen-binding domain.

Embodiment H213. The method of embodiment H211 or H212, wherein the antigen-binding domain comprises a scFv.

Embodiment H214. The method of any one of embodiments H185-H213, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD 137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CE AC AM5, a UL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF-b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD3, and a receptor for CD28.

Embodiment H215. The method of any one of embodiments H185-H213, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein.

Embodiment H216. The method of embodiment H215, wherein the soluble interleukin soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-l 7, IL-18, IL- 21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULPl6-binding protein.

Embodiment H217. The method of any one of embodiments H185-H213, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin or cytokine receptor.

Embodiment H218. The method of embodiment H217, wherein the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28. Embodiment H219. The method of any one of embodiments H166-H218, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide. Embodiment H220. The method of any one of embodiments H166-H218, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment H221. The method of any one of embodiments H166-H220, wherein the linker domain is a soluble tissue factor domain.

Embodiment H222. The method of embodiment H221, wherein the soluble tissue factor domain is a soluble human tissue factor domain. Embodiment H223. The method of embodiment H222, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1.

Embodiment H224. The method of embodiment H223, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment H225. The method of embodiment H224, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1.

Embodiment H226. The method of any one of embodiments H222-H225, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment H227. The method of embodiment H226, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. Embodiment H228. The method of any one of embodiments H221-H227, wherein the soluble tissue factor domain is not capable of binding to Factor Vila.

Embodiment H229. The method of any one of embodiments H221-H228, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment H230. The method of any one of embodiments H221-H229, wherein the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal. Embodiment H231. The method of any one of embodiments H221-H230, wherein the IgGl antibody construct comprises at least one antigen-binding domain that binds specifically to the soluble tissue factor domain.

Embodiment H232. The method of any one of embodiments H166-H220, wherein the linker domain is selected from the group consisting of: a kappa chain and a lambda chain.

Embodiment H233. The method of any one of embodiments H166-H232, wherein the IgGl antibody construct is a monoclonal IgGl antibody, where both antigen-binding domains in the monoclonal IgGl antibody bind specifically to the linker domain.

Embodiment H234. The method of any one of embodiments H166-H232, wherein the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen binding domains in the bispecific IgGl antibody binds specifically to the linker domain.

Embodiment H235. The method of any one of embodiments H166-H234, wherein the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (ILl5Ra) and a soluble IL-15. Embodiment H236. The method of embodiment H235, wherein the soluble IL15 has a D8N or D8A amino acid substitution. Embodiment H237. The method of embodiment H235 or H236, wherein the human ILl5Ra is a mature full-length ILl5Ra.

Embodiment H238. The method of any one of embodiments H166-H234, wherein the pair of affinity domains is selected from the group consisting of: barnase and barnstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. Embodiment H239. The method of any one of embodiments H166-H238, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminal end.

Embodiment H240. The method of any one of embodiments H166-H238, wherein the first chimeric polypeptide and/or the second chimeric polypeptide lacks a signal sequence at its N-terminal end.

Embodiment H241. The method of any one of embodiments H166-H240, wherein the contacting step is performed for a period of about 2 hours to about 20 days.

Embodiment H242. The method of embodiment H241, wherein the contacting step is performed for a period of about 1 day to about 15 days.

Embodiment H243. The method of any one of embodiments H166-H242, wherein the liquid culture medium is a serum-free liquid culture medium.

Embodiment H244. The method of any one of embodiments H166-H242, wherein the liquid culture medium is a chemically-defined liquid culture medium. Embodiment H245. The method of any one of embodiments H166-H244, wherein the liquid culture medium comprises the multi-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0.5: 1 to about 2: 1. Embodiment H246. The method of embodiment H245, wherein the liquid culture medium comprises the multi-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0.8: 1 to about 1.2: 1.

Embodiment H247. The method of any one of embodiments H166-H246, wherein the NK cell or T cell was previously obtained from a subject.

Embodiment H248. The method of embodiment H247, wherein the method further comprises obtaining the NK cell or T cell from the subject prior to the contacting step.

Embodiment H249. The method of any one of embodiments H166-H248, wherein the NK cell or T cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor. Embodiment H250. The method of any one of embodiments H166-H248, wherein the method further comprises, after the contacting step, introducing into the NK cell or the T cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Embodiment H251. The method of any one of embodiments H166-H248, wherein the method further comprises, before the contacting step, introducing into the NK cell or the T cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Embodiment H252. The method of any one of embodiments H166-H251, wherein the method further comprises, after the contacting step, isolating the NK cell or the T cell. Embodiment H253. The method of any one of embodiments H166-H252, wherein after the contacting step, the NK cell or the T cell has an increased level of expression or secretion of one or more proteins selected from the group consisting of: TNF-a, IFN-T, granzyme A, granzyme B, perforin, 2B4, CD8, CDl la, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, CDl58a, CDl58b, CDl58e, CD178, CD226, CD253, NKG2A, NKG2C, NKG2D, LIR-l, LILR-B1, KIR2DL1, KIR3DL1, KIR2DL2, KIR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-l, NKG2A, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-a, and IFN-g as compared to the level of expression or secretion of the one or more proteins prior to the contacting step.

Embodiment H254. The method of any one of embodiments H166-H253, wherein the method further comprises, after the contacting step, administering the NK cell or the T cell to a subject in need thereof.

Embodiment H255. The method of embodiment H254, wherein the subject has been identified or diagnosed as having an age-related disease or condition.

Embodiment H256. The method of embodiment H255, wherein the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. Embodiment H257. The method of embodiment H254, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment H258. The method of embodiment H257, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment H259. The method of embodiment H254, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment H260. The method of embodiment H259, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment H261. An activated NK cell or T cell produced by the method of any one of embodiments H166-H253.

Embodiment H262. A pharmaceutical composition comprising the activated NK cell or the activated T cell of embodiment H261. Embodiment H263. A kit comprising a pharmaceutical composition comprising the activated NK cell or the activated T cell of embodiment H261.

Embodiment H264. A method of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the activated NK cell or the activated T cell of embodiment H261 or the pharmaceutical composition of embodiment H262.

Embodiment H265. The method of embodiment H264, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment H266. The method of embodiment H265, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment H267. The method of embodiment H264, wherein the subject has been identified or diagnosed as having an aging-related disease or condition.

Embodiment H268. The method of embodiment H267, wherein the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction

Embodiment H269. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the activated NK cell or the activated T cell of embodiment H261 or the pharmaceutical composition of embodiment H262.

Embodiment H270. The method of embodiment H269, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment H271. The method of embodiment H270, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment H272. The method of embodiment H269, wherein the subject has been identified or diagnosed as having an aging-related disease or condition. Embodiment H273. The method of embodiment H272, wherein the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment H274. The method of embodiment H269, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment H275. The method of embodiment H274, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment H276. A kit comprising:

(1) a multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a linker domain; and

(iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and

(2) an effective amount of an IgGl antibody that comprises at least one antigen-binding domain that binds specifically to the linker domain.

Embodiment H277. The kit of embodiment H276, wherein the first target-binding domain and the linker domain directly abut each other in the first chimeric polypeptide. Embodiment H278. The kit of embodiment H276, wherein the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the linker domain in the first chimeric polypeptide.

Embodiment H279. The kit of any one of embodiments H276-H278, wherein the linker domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide.

Embodiment H280. The kit of any one of embodiments H276-H278, wherein the first chimeric polypeptide further comprises a linker sequence between the linker domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H281. The kit of any one of embodiments H276-H280, wherein the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide.

Embodiment H282. The kit of any one of embodiments H276-H280, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide. Embodiment H283. The kit of any one of embodiments H276-H282, wherein the first target-binding domain and the second target-binding domain bind specifically to the same antigen. Embodiment H284. The kit of embodiment H283, wherein the first target-binding domain and the second target-binding domain bind specifically to the same epitope.

Embodiment H285. The kit of embodiment H284, wherein the first target-binding domain and the second target-binding domain comprise the same amino acid sequence.

Embodiment H286. The kit of any one of embodiments H276-H282, wherein the first target-binding domain and the second target-binding domain bind specifically to different antigens. Embodiment H287. The kit of any one of embodiments H276-H286, wherein one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain.

Embodiment H288. The kit of embodiment H287, wherein the first target-binding domain and the second target-binding domain are each antigen-binding domains.

Embodiment H289. The kit of embodiment H287 or H288, wherein the antigen binding domain comprises a scFv or a single domain antibody. Embodiment H290. The kit of any one of embodiments H276-H289, wherein one or both of the first target-binding domain and the second target-binding domain bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD 19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3,

CD272, VISTA, CD137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P- cadherin, CEACAM5, a EIL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD 122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF- b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-l 5, a receptor for IL- 17, a receptor for IL-l 8, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.

Embodiment H291. The kit of any one of embodiments H276-H289, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein.

Embodiment H292. The kit of embodiment H291, wherein the soluble interleukin or cytokine protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-l 2, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment H293. The kit of any one of embodiments H276-H289, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin or cytokine receptor.

Embodiment H294. The kit of embodiment H293, wherein the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD 155, or a soluble CD28. Embodiment H295. The kit of any one of embodiments H276-H294, wherein the first chimeric polypeptide further comprises one or more additional target-binding domain(s), where at least one of the one or more target-binding domain(s) is positioned between the linker domain and the first domain of the pair of affinity domains.

Embodiment H296. The kit of embodiment H295, wherein the first chimeric polypeptide further comprises a linker sequence between the linker domain and the at least one of the one or more target antigen-binding domain(s), and/or a linker sequence between the at least one of the one or more target antigen-binding domain(s) and the first domain of the pair of affinity domains.

Embodiment H297. The kit of any one of embodiments H276-H294, wherein the first chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide.

Embodiment H298. The kit of embodiment H297, wherein at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment H299. The kit of embodiment H297, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains. Embodiment H300. The kit of embodiment H297, wherein the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment H301. The kit of embodiment H297, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first target-binding domain. Embodiment H302. The kit of embodiment H297, wherein at least one of the one or more additional target-binding domains is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains is positioned between the linker domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H303. The kit of embodiment H302, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H304. The kit of embodiment H302, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H305. The kit of embodiment H302, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H306. The kit of embodiment H302, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H307. The kit of embodiment H302, wherein the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains, directly abuts the linker domain and/or the first domain of the pair of affinity domains. Embodiment H308. The kit of embodiment H302, wherein the first chimeric polypeptide further comprises a linker sequence disposed (i) between the linker domain and the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains.

Embodiment H309. The kit of any one of embodiments H276-H308, wherein the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment H310. The kit of embodiment H309, wherein at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment H311. The kit of embodiment H309, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment H312. The kit of embodiment H309, wherein at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide.

Embodiment H313. The kit of embodiment H309, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second target-binding domain in the second chimeric polypeptide. Embodiment H314. The kit of any one of embodiments H295-H313, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. Embodiment H315. The kit of embodiment H314, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope.

Embodiment H316. The kit of embodiment H315, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

Embodiment H317. The kit of embodiment H314, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same antigen.

Embodiment H318. The kit of embodiment H317, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same epitope.

Embodiment H319. The kit of embodiment H318, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each comprise the same amino acid sequence. Embodiment H320. The kit of any one of embodiments H295-H313, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to different antigens.

Embodiment H321. The kit of any one of embodiments H295-H320, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen-binding domain. Embodiment H322. The kit of embodiment H321, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen-binding domain. Embodiment H323. The kit of embodiment H321 or H322, wherein the antigen binding domain comprises a scFv.

Embodiment H324. The kit of any one of embodiments H295-H323, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD 137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2,

HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CE AC AM5, a UL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-PRII), a ligand of TGF-b receptor III (TGF^RIII), a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-l 2, a receptor for IL-l 5, a receptor for IL-l 7, a receptor for IL-l 8, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD3, and a receptor for CD28.

Embodiment H325. The kit of any one of embodiments H295-H323, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. Embodiment H326. The kit of embodiment H325, wherein the soluble interleukin or cytokine protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-l 2, IL-15, IL-l 7, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a EILP16-binding protein.

Embodiment H327. The kit of any one of embodiments H295-H323, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin or cytokine receptor.

Embodiment H328. The kit of embodiment H327, wherein the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28.

Embodiment H329. The kit of any one of embodiments H295-H328, wherein one or both of the first target-binding domain and the second target-binding domain is a ligand of a co-stimulatory molecule.

Embodiment H330. The kit of embodiment H329, wherein the ligand of a co- stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l.

Embodiment H331. The kit of any one of embodiments H276-H330, wherein the linker domain is a soluble tissue factor domain.

Embodiment H332. The kit of embodiment H331, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment H333. The kit of embodiment H332, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1. Embodiment H334. The kit of embodiment H333, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment H335. The kit of embodiment H334, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1.

Embodiment H336. The kit of any one of embodiments H332-H335, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment H337. The kit of embodiment H336, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. Embodiment H338. The kit of any one of embodiments H331-H337, wherein the soluble tissue factor domain is not capable of binding to Factor Vila.

Embodiment H339. The kit of any one of embodiments H331-H338, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment H340. The kit of any one of embodiments H331-H339, wherein the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

Embodiment H341. The kit of any one of embodiments H331-H340, wherein the IgGl antibody construct comprises at least one antigen-binding domain that binds specifically to the soluble tissue factor domain.

Embodiment H342. The kit of any one of embodiments H276-H330, wherein the linker domain is selected from the group consisting of: a kappa chain and a lambda chain.

Embodiment H343. The kit of any one of embodiments H276-H342, wherein the IgGl antibody construct is a monoclonal IgGl antibody, where both antigen-binding domains in the monoclonal IgGl antibody bind specifically to the linker domain. Embodiment H344. The kit of any one of embodiments H276-H342, wherein the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen binding domains in the bispecific IgGl antibody binds specifically to the linker domain. Embodiment H345. The kit of any one of embodiments H276-H344, wherein the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (ILl5Ra) and a soluble IL-15.

Embodiment H346. The kit of embodiment H345, wherein the soluble IL15 has a D8N or D8A amino acid substitution.

Embodiment H347. The kit of embodiment H345 or H346, wherein the human ILl5Ra is a mature full-length ILl5Ra. Embodiment H348. The kit of any one of embodiments H276-H344, wherein the pair of affinity domains is selected from the group consisting of: barnase and barnstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

Embodiment H349. The kit of any one of embodiments H276-H348, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminal end. Embodiment H350. The kit of any one of embodiments H276-H348, wherein the first chimeric polypeptide and/or the second chimeric polypeptide lacks a signal sequence at its N-terminal end.

Embodiment H351. The method of any one of embodiments H1-H16, wherein one or both of the first target-binding domain and the second target-binding domain is a ligand of a co-stimulatory molecule. Embodiment H352. The method of embodiment H351, wherein the ligand of a co- stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l. Embodiment H353. The method of any one of embodiments H1-H62, wherein the single-chain chimeric polypeptide further comprises a signal sequence at its N-terminal end.

Embodiment H354. The method of any one of embodiments H1-H62, wherein the single-chain chimeric polypeptide lacks a signal sequence at its N-terminal end.

Embodiment H355. The kit of any one of embodiments H101-H162, wherein the single-chain chimeric polypeptide further comprises a signal sequence at its N-terminal end.

Embodiment H356. The kit of any one of embodiments H101-H162, wherein the single-chain chimeric polypeptide lacks a signal sequence at its N-terminal end.

Embodiment H357. The method of any one of embodiments Ell 66-Ell 79, wherein one or both of the first target-binding domain and the second target-binding domain is a ligand of a co-stimulatory molecule.

Embodiment E1358. The method of embodiment E1357, wherein the ligand of a co- stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l.

Embodiment H359. The method of any one of embodiments H185-H217, wherein one or both of the first target-binding domain and the second target-binding domain is a ligand of a co-stimulatory molecule. Embodiment H360. The method of embodiment H359, wherein the ligand of a co- stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l. Embodiment H361. The kit of any one of embodiments H276-H294, wherein one or both of the first target-binding domain and the second target-binding domain is a ligand of a co-stimulatory molecule.

Embodiment H362. The kit of embodiment H361 wherein the ligand of a co- stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l.

Embodiment H363. The kit of any one of embodiments H276-H330, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C- terminal end of the first chimeric polypeptide.

Embodiment H364. The kit of any one of embodiments H276-H328, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment H365. A method of increasing the glucose consumption of an immune cell, the method comprising:

contacting an immune cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and optionally (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for glucose consumption in the immune cell,

wherein the first target-binding domain and the second target-binding domain are each independently selected from the group consisting of: a soluble interleukin or cytokine protein, an antigen-binding domain, a soluble interleukin or cytokine receptor, and ligands of co-stimulatory molecules.

Embodiment H366. A method of increasing the oxidative phosphorylation of an immune cell, the method comprising:

contacting an immune cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and optionally (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for oxidative

phosphorylation in the immune cell,

wherein the first target-binding domain and the second target-binding domain are each independently selected from the group consisting of: a soluble interleukin or cytokine protein, an antigen-binding domain, a soluble interleukin or cytokine receptor, and ligands of co-stimulatory molecules.

Embodiment H367. A method of increasing the aerobic glycolysis of an immune cell, the method comprising:

contacting an immune cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and optionally (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for aerobic glycolysis in the immune cell,

wherein the first target-binding domain and the second target-binding domain are each independently selected from the group consisting of: a soluble interleukin or cytokine protein, an antigen-binding domain, a soluble interleukin or cytokine receptor, and ligands of co-stimulatory molecules. Embodiment H368. A method of increasing the extracellular acidification rate

(ECAR) of an immune cell, the method comprising: contacting an immune cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and optionally (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for extracellular acidification by the immune cell,

wherein the first target-binding domain and the second target-binding domain are each independently selected from the group consisting of: a soluble interleukin or cytokine protein, an antigen-binding domain, a soluble interleukin or cytokine receptor, and ligands of co-stimulatory molecules.

Embodiment H369. A method of increasing the mitochondrial oxygen

consumption rate of an immune cell, the method comprising:

contacting an immune cell in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and optionally (ii) an IgGl antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for mitochondrial oxygen consumption rate by the immune cell,

wherein the first target-binding domain and the second target-binding domain are each independently selected from the group consisting of: a soluble interleukin or cytokine protein, an antigen-binding domain, a soluble interleukin or cytokine receptor, and ligands of co-stimulatory molecules. Embodiment H370. The method of any one of embodiments H365-H369, wherein the liquid culture medium comprises the single-chain chimeric polypeptide and the IgGl antibody construct.

Embodiment H371. The method of embodiment H370, wherein the IgGl antibody construct is a monoclonal IgGl antibody, where both of the antigen-binding domains in the monoclonal IgGl antibody bind specifically to the linker domain. Embodiment H372. The method of embodiment H370, wherein the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the linker domain. Embodiment H373. The method of any one of embodiments H365-H372, wherein the contacting step is performed for a period of about 2 hours to about 20 days.

Embodiment H374. The method of embodiment H373, wherein the contacting step is performed for a period of about 1 day to about 15 days.

Embodiment H375. The method of any one of embodiments H365-H374, wherein the liquid culture medium is a serum-free liquid culture medium.

Embodiment H376. The method of any one of embodiments H365-H374, wherein the liquid culture medium is a chemically-defined liquid culture medium.

Embodiment H377. The method of any one of embodiments H365-H374, wherein the liquid culture medium comprises serum. Embodiment H378. The method of any one of embodiments H365-H377, wherein the liquid culture medium comprises the single-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0.5: 1 to about 2: 1.

Embodiment H379. The method of embodiment H378, wherein the liquid culture medium comprises the single-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0.8:1 to about 1.2: 1.

Embodiment H380. The method of any one of embodiments H365-H379, wherein the first target-binding domain and the linker domain directly abut each other. Embodiment H381. The method of any one of embodiments H365-H379, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the linker domain. Embodiment H382. The method of any one of embodiments H365-H381, wherein the linker domain and the second target-binding domain directly abut each other.

Embodiment H383. The method of any one of embodiments H365-H381, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the linker domain and the second target-binding domain.

Embodiment H384. The method of any one of embodiments H365-H379, wherein the first target-binding domain and the second target-binding domain directly abut each other.

Embodiment H385. The method of any one of embodiments H365-H379, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the second target-binding domain. Embodiment H386. The method of embodiment H384 or H385, wherein the second target-binding domain and the linker domain directly abut each other.

Embodiment H387. The method of embodiment H384 or H385, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the second target-binding domain and the linker domain.

Embodiment H388. The method of any one of embodiments H365-H387, wherein the first target-binding domain and the second target-binding domain bind specifically to the same antigen. Embodiment H389. The method of embodiment H388, wherein the first target- binding domain and the second target-binding domain bind specifically to the same epitope. Embodiment H390. The method of embodiment H389, wherein the first target- binding domain and the second target-binding domain comprise the same amino acid sequence.

Embodiment H391. The method of any one of embodiments H365-H387, wherein the first target-binding domain and the second target-binding domain bind specifically to different antigens.

Embodiment H392. The method of any one of embodiments H365-H391, wherein one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain.

Embodiment H393. The method of embodiment H392, wherein the first target- binding domain and the second target-binding domain are each an antigen-binding domain.

Embodiment H394. The method of embodiment H392 or H393, wherein antigen binding domain comprises a scFv or a single domain antibody.

Embodiment H395. The method of any one of embodiments H365-H394, wherein one or both of the first target-binding domain and the second target-binding domain bind to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20,

CD 19, CD22, CD52, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC,

Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P- cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD 122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-pRII), a ligand of TGF- PRIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-l 5, a receptor for IL- 17, a receptor for IL-l 8, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.

Embodiment H396. The method of any one of embodiments H365-H394, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein.

Embodiment H397. The method of embodiment H396, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL- 21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment H398. The method of any one of embodiments H365-H394, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin or cytokine receptor. Embodiment H399. The method of embodiment H398, wherein the soluble interleukin or cytokine receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28. Embodiment H400. The method of any one of embodiments H365-H399, wherein the linker domain is a soluble tissue factor domain. Embodiment H401. The method of embodiment H400, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment H402. The method of embodiment H401, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1.

Embodiment H403. The method of embodiment H402, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment H404. The method of embodiment H403, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1.

Embodiment H405. The method of any one of embodiments H401-H404, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. Embodiment H406. The method of embodiment H405, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment H407. The method of any one of embodiments H400-H406, wherein the soluble tissue factor domain is not capable of binding Factor Vila.

Embodiment H408. The method of any one of embodiments H400-H407, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment H409. The method of any one of embodiments H400-H408, wherein the single-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

Embodiment H410. The method of any one of embodiments H400-H409, wherein the IgGl antibody construct comprises at least one antigen-binding domain that binds specifically to the soluble tissue factor domain. Embodiment H411. The method of any one of embodiments H365-H399, wherein the linker domain is selected from the group consisting of: a kappa chain and a lambda chain.

Embodiment H412. The method of any one of embodiments H365-H411, wherein the IgGl antibody construct is a monoclonal IgGl antibody, where both of the antigen binding domains in the monoclonal IgGl antibody bind specifically to the linker domain.

Embodiment H413. The method of any one of embodiments H365-H411, wherein the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen- binding domains in the bispecific IgGl antibody binds specifically to the linker domain.

Embodiment H414. The method of any one of embodiments H365-H413, wherein the single-chain chimeric polypeptide further comprises one or more additional target binding domains at its N- and/or C-terminus.

Embodiment H415. The method of embodiment H414, wherein the single-chain chimeric polypeptide comprises one or more additional target-binding domains at its N- terminus. Embodiment H416. The method of embodiment H415, wherein one or more additional target-binding domains directly abuts the first target-binding domain, the second target-binding domain, or the linker domain.

Embodiment H417. The method of embodiment H416, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target-binding domains and the first target-binding domain, the second target binding domain, or the linker domain.

Embodiment H418. The method of embodiment H414, wherein the single-chain chimeric polypeptide comprises one or more additional target-binding domains at its C- terminus. Embodiment H419. The method of embodiment H418, wherein one of the one or more additional target-binding domains directly abuts the first target-binding domain, the second target-binding domain, or the linker domain. Embodiment H420. The method of embodiment H418, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target-binding domains and the first target-binding domain, the second target- binding domain, or the linker domain. Embodiment H421. The method of embodiment H414, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N- terminus and the C-terminus.

Embodiment H422. The method of embodiment H421, wherein one of the one or more additional target-binding domains at the N-terminus directly abuts the first target- binding domain, the second target-binding domain, or the linker domain.

Embodiment H423. The method of embodiment H421, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional target-binding domains at the N-terminus and the first target-binding domain, the second target-binding domain, or the linker domain.

Embodiment H424. The method of embodiment H421, wherein one of the one or more additional target-binding domains at the C-terminus directly abuts the first target- binding domain, the second target-binding domain, or the linker domain.

Embodiment H425. The method of embodiment H421, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional target-binding domains at the C-terminus and the first target-binding domain, the second target-binding domain, or the linker domain. Embodiment H426. The method of any one of embodiments H414-H425, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. Embodiment H427. The method of embodiment H426, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope.

Embodiment H428. The method of embodiment H427, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

Embodiment H429. The method of embodiment H426, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains each bind specifically to the same antigen.

Embodiment H430. The method of embodiment H429, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains each bind specifically to the same epitope.

Embodiment H431. The method of embodiment H430, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains each comprise the same amino acid sequence. Embodiment H432. The method of any one of embodiments H414-H425, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to different antigens.

Embodiment H433. The method of any one of embodiments H414-H432, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen-binding domain. Embodiment H434. The method of embodiment H433, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains are each an antigen-binding domain. Embodiment H435. The method of embodiment H433 or H434, wherein the antigen-binding domain comprises a scFv or a single domain antibody.

Embodiment H436. The method of any one of embodiments H414-H435, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD 137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2,

HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CE AC AM5, a UL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-PRII), a ligand of TGF-PRIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD28.

Embodiment H437. The method of any one of embodiments H414-H432, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein. Embodiment H438. The method of embodiment H437, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL- 21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULPl6-binding protein.

Embodiment H439. The method of any one of embodiments H414-H432, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin or cytokine receptor.

Embodiment H440. The method of embodiment H439, wherein the soluble interleukin or cytokine receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28.

Embodiment H441. The method of any one of embodiments H414-H432, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a ligand of a co-stimulatory molecule.

Embodiment H442. The method of embodiment H441, wherein the ligand of a co- stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-l.

Embodiment H443. The method of any one of embodiments H365-H442, wherein the single-chain chimeric polypeptide further comprises a peptide tag positioned at the N- terminal end or the C-terminal end of the single-chain chimeric polypeptide.

Embodiment H444. The method of any one of embodiments H365-H443, wherein the NK cell or T cell was previously obtained from a subject. Embodiment H445. The method of embodiment H444, wherein the method further comprises obtaining the NK cell or T cell from the subject prior to the contacting step. Embodiment H446. The method of any one of embodiments H365-H445, wherein the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

Embodiment H447. The method of any one of embodiments H365-H445, wherein the method further comprises, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Embodiment H448. The method of any one of embodiments H365-H445, wherein the method further comprises, before the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Embodiment H449. The method of any one of embodiments H365-H448, wherein the method further comprises, after the contacting step, isolating the immune cell. Embodiment H450. The method of any one of embodiments H365-H449, wherein after the contacting step, the immune cell has an increased level of expression or secretion of one or more proteins selected from the group consisting of: TNF-a, IFN-T, granzyme A, granzyme B, perforin, 2B4, CD8, CDl la, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, CDl58a, CDl58b, CDl58e, CD178, CD226, CD253, NKG2A, NKG2C, NKG2D, LIR-l, LILR-B1,

KIR2DL1, KIR3DL1, KIR2DL2, KIR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-l, NKG2A, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-a, and IFN-g as compared to the level of expression or secretion of the one or more proteins prior to the contacting step. Embodiment H451. The method of any one of embodiments H365-H450, wherein the method further comprises, after the contacting step, administering the immune cell to a subject in need thereof.

Embodiment H452. The method of embodiment H451, wherein the subject has been identified or diagnosed as having an age-related disease or condition.

Embodiment H453. The method of embodiment H452, wherein the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment H454. The method of embodiment H451, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment H455. The method of embodiment H454, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment H456. The method of embodiment H451, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment H457. The method of embodiment H456, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment H458. An activated immune cell produced by the method of any one of embodiments H365-H450.

Embodiment H459. A pharmaceutical composition comprising the activated immune cell of embodiment H458.

Embodiment H460. A kit comprising a pharmaceutical composition comprising the activated immune cell of embodiment H458.

Embodiment H461. A method of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the activated immune cell of embodiment H458 or the pharmaceutical composition of embodiment H459.

Embodiment H462. The method of embodiment H461, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment H463. The method of embodiment H462, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment H464. The method of embodiment H462, wherein the subject has been identified or diagnosed as having an aging-related disease or condition.

Embodiment H465. The method of embodiment H464, wherein the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment H466. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the activated immune cell of embodiment H458 or the pharmaceutical composition of embodiment H459. Embodiment H467. The method of embodiment H466, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment H468. The method of embodiment H467, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment H469. The method of embodiment H466, wherein the subject has been identified or diagnosed as having an aging-related disease or condition.

Embodiment H470. The method of embodiment H469, wherein the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. Embodiment H471. The method of embodiment H466, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment H472. The method of embodiment H471, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, or influenza virus.

Embodiment H473. A method of increasing the glucose consumption of an immune cell, the method comprising:

contacting an immune cell in a liquid culture medium comprising (1) an effective amount of a multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a linker domain; and

(iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein the first chimeric polypeptide and the second chimeric

polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and

(2) an effective amount of an IgGl antibody construct comprising at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for glucose consumption in the immune cell.

Embodiment H474. A method of increasing the oxidative phosphorylation of an immune cell, the method comprising:

contacting an immune cell in a liquid culture medium comprising (1) an effective amount of a multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising: (i) a first target-binding domain;

(ii) a linker domain; and

(iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein the first chimeric polypeptide and the second chimeric

polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and

(2) an effective amount of an IgGl antibody construct comprising at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for oxidative phosphorylation in the immune cell.

Embodiment H475. A method of increasing the aerobic glycolysis of an immune cell, the method comprising:

contacting an immune cell in a liquid culture medium comprising (1) an effective amount of a multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a linker domain; and

(iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein the first chimeric polypeptide and the second chimeric

polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and

(2) an effective amount of an IgGl antibody construct comprising at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for aerobic glycolysis in the immune cell. Embodiment H476. A method of increasing the extracellular acidification rate (ECAR) of an immune cell, the method comprising:

contacting an immune cell in a liquid culture medium comprising (1) an effective amount of a multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a linker domain; and

(iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein the first chimeric polypeptide and the second chimeric

polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and

(2) an effective amount of an IgGl antibody construct comprising at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for extracellular acidification by the immune cell.

Embodiment H477. A method of increasing the mitochondrial oxygen

consumption rate of an immune cell, the method comprising:

contacting an immune cell in a liquid culture medium comprising (1) an effective amount of a multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a linker domain; and

(iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric

polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and

(2) an effective amount of an IgGl antibody construct comprising at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for mitochondrial oxygen consumption rate by the immune cell.

Embodiment H478. The method of any one of embodiments H473-H477, wherein the liquid culture medium comprises the single-chain chimeric polypeptide and the IgGl antibody construct.

Embodiment H479. The method of embodiment H478, wherein the IgGl antibody construct is a monoclonal IgGl antibody, where both of the antigen-binding domains in the monoclonal IgGl antibody bind specifically to the linker domain.

Embodiment H480. The method of embodiment H478, wherein the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen-binding domains in the bispecific IgGl antibody binds specifically to the linker domain. Embodiment H481. The method of any one of embodiments H473-H480, wherein the contacting step is performed for a period of about 2 hours to about 20 days.

Embodiment H482. The method of embodiment H481, wherein the contacting step is performed for a period of about 1 day to about 15 days.

Embodiment H483. The method of any one of embodiments H473-H482, wherein the liquid culture medium is a serum-free liquid culture medium.

Embodiment H484. The method of any one of embodiments H473-H482, wherein the liquid culture medium is a chemically-defined liquid culture medium. Embodiment H485. The method of any one of embodiments H473-H482, wherein the liquid culture medium comprises serum.

Embodiment H486. The method of any one of embodiments H473-H485, wherein the liquid culture medium comprises the single-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0.5: 1 to about 2: 1.

Embodiment H487. The method of embodiment H486, wherein the liquid culture medium comprises the single-chain chimeric polypeptide and the IgGl antibody construct at a molar ratio of about 0.8:1 to about 1.2: 1.

Embodiment H488. The method of any one of embodiments H473-H487, wherein the first target-binding domain and the linker domain directly abut each other in the first chimeric polypeptide.

Embodiment H489. The method of any one of embodiments H473-H487, wherein the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the linker domain in the first chimeric polypeptide. Embodiment H490. The method of any one of embodiments H473-H489, wherein the linker domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide.

Embodiment H491. The method of any one of embodiments H473-H489, wherein the first chimeric polypeptide further comprises a linker sequence between the linker domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H492. The method of any one of embodiments H473-H491, wherein the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. Embodiment H493. The method of any one of embodiments H473-H491, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

Embodiment H494. The method of any one of embodiments H473-H493, wherein the first target-binding domain and the second target-binding domain bind specifically to the same antigen. Embodiment H495. The method of embodiment H494, wherein the first target- binding domain and the second target-binding domain bind specifically to the same epitope.

Embodiment H496. The method of embodiment H495, wherein the first target- binding domain and the second target-binding domain comprise the same amino acid sequence.

Embodiment H497. The method of any one of embodiments H473-H493, wherein the first target-binding domain and the second target-binding domain bind specifically to different antigens.

Embodiment H498. The method of any one of embodiments H473-H497, wherein one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain.

Embodiment H499. The method of embodiment H498, wherein the first target- binding domain and the second target-binding domain are each antigen-binding domains.

Embodiment H500. The method of embodiment H498 or H499, wherein the antigen-binding domain comprises a scFv or a single domain antibody. Embodiment H501. The method of any one of embodiments H473-H500, wherein one or both of the first target-binding domain and the second target-binding domain bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD 19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P- cadherin, CEACAM5, a EIL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD 122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF- b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-l 5, a receptor for IL- 17, a receptor for IL-l 8, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28. Embodiment H502. The method of any one of embodiments H473-H497, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein.

Embodiment H503. The method of embodiment H502, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL- 21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment H504. The method of any one of embodiments H473-H497, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin or cytokine receptor. Embodiment H505. The method of embodiment H504, wherein the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28.

Embodiment H506. The method of any one of embodiments H473-H505, wherein the first chimeric polypeptide further comprises one or more additional target-binding domain(s), where at least one of the one or more target-binding domain(s) is positioned between the linker domain and the first domain of the pair of affinity domains.

Embodiment H507. The method of embodiment H506, wherein the first chimeric polypeptide further comprises a linker sequence between the linker domain and the at least one of the one or more target antigen-binding domain(s), and/or a linker sequence between the at least one of the one or more target antigen-binding domain(s) and the first domain of the pair of affinity domains.

Embodiment H508. The method of any one of embodiments H473-H505, wherein the first chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide.

Embodiment H509. The method of embodiment H508, wherein at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment H510. The method of embodiment H508, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains. Embodiment H511. The method of embodiment H508, wherein the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide. Embodiment H512. The method of embodiment H508, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first target-binding domain.

Embodiment H513. The method of embodiment H508, wherein at least one of the one or more additional target-binding domains is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target- binding domains is positioned between the linker domain and the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment H514. The method of embodiment H513, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment H515. The method of embodiment H513, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment H516. The method of embodiment H513, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. Embodiment H517. The method of embodiment H513, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H518. The method of embodiment H513, wherein the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains, directly abuts the linker domain and/or the first domain of the pair of affinity domains.

Embodiment H519. The method of embodiment H513, wherein the first chimeric polypeptide further comprises a linker sequence disposed (i) between the linker domain and the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target-binding domains positioned between the linker domain and the first domain of the pair of affinity domains.

Embodiment H520. The method of any one of embodiments H473-H519, wherein the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end and/or the C-terminal end of the second chimeric polypeptide.

Embodiment H521. The method of embodiment H520, wherein at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment H522. The method of embodiment H520, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide. Embodiment H523. The method of embodiment H520, wherein at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide. Embodiment H524. The method of embodiment H520, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second target-binding domain in the second chimeric polypeptide. Embodiment H525. The method of any one of embodiments H506-H524, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen.

Embodiment H526. The method of embodiment H525, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope.

Embodiment H527. The method of embodiment H526, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

Embodiment H528. The method of embodiment H525, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains each bind specifically to the same antigen.

Embodiment H529. The method of embodiment H528, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains each bind specifically to the same epitope. Embodiment H530. The method of embodiment H529, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains each comprise the same amino acid sequence.

Embodiment H531. The method of any one of embodiments H506-H524, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to different antigens.

Embodiment H532. The method of any one of embodiments H506-H531, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen-binding domain.

Embodiment H533. The method of embodiment H532, wherein the first target- binding domain, the second target-binding domain, and the one or more additional target- binding domains are each an antigen-binding domain.

Embodiment H534. The method of embodiment H532 or H533, wherein the antigen-binding domain comprises a scFv.

Embodiment H535. The method of any one of embodiments H506-H534, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group consisting of: CDl6a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-l, VEGF, IL-6R, IL-4, IL-10, PDL-l, TIGIT, PD-l, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD 137, CD40, CD47, CD70, 0X40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CE AC AM5, a UL16-binding protein, HLA-DR, DLL4, TYR03, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-b receptor II (TGF-b RII), a ligand of TGF-b RIII, a ligand of DNAM-l, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-l, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD3, and a receptor for CD28.

Embodiment H536. The method of any one of embodiments H506-H534, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin, soluble cytokine protein, or soluble cell surface protein.

Embodiment H537. The method of embodiment H536, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of: IL-l, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-l 7, IL-18, IL- 21, PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULPl6-binding protein.

Embodiment H538. The method of any one of embodiments H506-H534, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin or cytokine receptor.

Embodiment H539. The method of embodiment H538, wherein the soluble receptor is a soluble TGF-b receptor II (TGF-b RII), a soluble TGF-b RIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-l, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28. Embodiment H540. The method of any one of embodiments H473-H539, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide. Embodiment H541. The method of any one of embodiments H473-H539, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment H542. The method of any one of embodiments H473-H541, wherein the linker domain is a soluble tissue factor domain.

Embodiment H543. The method of embodiment H542, wherein the soluble tissue factor domain is a soluble human tissue factor domain. Embodiment H544. The method of embodiment H543, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1.

Embodiment H545. The method of embodiment H544, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment H546. The method of embodiment H545, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1.

Embodiment H547. The method of any one of embodiments H543-H546, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment H548. The method of embodiment H547, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. Embodiment H549. The method of any one of embodiments H542-H548, wherein the soluble tissue factor domain is not capable of binding to Factor Vila.

Embodiment H550. The method of any one of embodiments H542-H549, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment H551. The method of any one of embodiments H542-H550, wherein the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal. Embodiment H552. The method of any one of embodiments H542-H551, wherein the IgGl antibody construct comprises at least one antigen-binding domain that binds specifically to the soluble tissue factor domain.

Embodiment H553. The method of any one of embodiments H473-H541, wherein the linker domain is selected from the group consisting of: a kappa chain and a lambda chain.

Embodiment H554. The method of any one of embodiments H473-H553, wherein the IgGl antibody construct is a monoclonal IgGl antibody, where both antigen-binding domains in the monoclonal IgGl antibody bind specifically to the linker domain.

Embodiment H555. The method of any one of embodiments H473-H553, wherein the IgGl antibody construct is a bispecific IgGl antibody, where one of the two antigen binding domains in the bispecific IgGl antibody binds specifically to the linker domain.

Embodiment H556. The method of any one of embodiments H473-H555, wherein the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (ILl5Ra) and a soluble IL-15. Embodiment H557. The method of embodiment H556, wherein the soluble IL15 has a D8N or D8A amino acid substitution. Embodiment H558. The method of embodiment H556 or H557, wherein the human ILl5Ra is a mature full-length ILl5Ra.

Embodiment H559. The method of any one of embodiments H473-H555, wherein the pair of affinity domains is selected from the group consisting of: barnase and barnstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. Embodiment H560. The method of any one of embodiments H473-H559, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminal end.

Embodiment H561. The method of any one of embodiments H473-H559, wherein the first chimeric polypeptide and/or the second chimeric polypeptide lacks a signal sequence at its N-terminal end.

Embodiment H562. The method of any one of embodiments H473-H561, wherein the immune cell was previously obtained from a subject.

Embodiment H563. The method of embodiment H562, wherein the method further comprises obtaining the immune cell from the subject prior to the contacting step.

Embodiment H564. The method of any one of embodiments H473-H563, wherein the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

Embodiment H565. The method of any one of embodiments H473-H563, wherein the method further comprises, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Embodiment H566. The method of any one of embodiments H473-H563, wherein the method further comprises, before the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Embodiment H567. The method of any one of embodiments H473-H566, wherein the method further comprises, after the contacting step, isolating the immune cell.

Embodiment H568. The method of any one of embodiments H473-H567, wherein after the contacting step, the immune cell has an increased level of expression or secretion of one or more proteins selected from the group consisting of: TNF-a, IFN-T, granzyme A, granzyme B, perforin, 2B4, CD8, CDl la, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, CDl58a, CDl58b, CDl58e, CD178, CD226, CD253, NKG2A, NKG2C, NKG2D, LIR-l, LILR-B1, KIR2DL1, KIR3DL1, KIR2DL2, KIR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-l, NKG2A, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-a, and IFN-g as compared to the level of expression or secretion of the one or more proteins prior to the contacting step.

Embodiment H569. The method of any one of embodiments H473-H568, wherein the method further comprises, after the contacting step, administering the immune cell to a subject in need thereof.

Embodiment H570. The method of embodiment H569, wherein the subject has been identified or diagnosed as having an age-related disease or condition.

Embodiment H571. The method of embodiment H570, wherein the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment H572. The method of embodiment H569, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment H573. The method of embodiment H572, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment H574. The method of embodiment H569, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment H575. The method of embodiment H574, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus. Embodiment H576. An activated immune cell produced by the method of any one of embodiments H473-H568.

Embodiment H577. A pharmaceutical composition comprising the activated cell of embodiment H576.

Embodiment H578. A kit comprising a pharmaceutical composition comprising the activated immune cell of embodiment H261.

Embodiment H579. A method of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the activated immune cell of embodiment H576 or the pharmaceutical composition of embodiment H577.

Embodiment H580. The method of embodiment H579, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment H581. The method of embodiment H580, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. Embodiment H582. The method of embodiment H579, wherein the subject has been identified or diagnosed as having an aging-related disease or condition.

Embodiment H583. The method of embodiment H582, wherein the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction

Embodiment H584. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the activated immune cell of embodiment H576 or the pharmaceutical composition of embodiment H577.

Embodiment H585. The method of embodiment H584, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment H586. The method of embodiment H585, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastic and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment H587. The method of embodiment H584, wherein the subject has been identified or diagnosed as having an aging-related disease or condition.

Embodiment H588. The method of embodiment H587, wherein the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia,

glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment H589. The method of embodiment H584, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment H590. The method of embodiment H589, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.