COMPOSITIONS AND METHODS FOR

TCR REPROGRAMMING USING INDUCIBLE FUSION PROTEINS

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/671,342, filed May 14, 2018, which is entirely incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Engineered T cells expressing either T Cell Receptor Fusion Constructs (TRuC™s) or chimeric antigen receptors (CARs) at their surface have produced exciting anti-tumor responses in vitro and in vivo. Although studies have demonstrated the potential of these technologies, there have been some issues that have raised concerns associated with "on-target off-tumor" effect, especially in healthy tissues which may express low levels of the targeted antigen. As such the ability to control the functional activity of CAR or TruC™ T cells is an important objective to ensure optimal safety and efficacy of engineered T cell therapies in solid organ tumors. As such strategies that may improve engineered T cell discrimination between healthy tissue and cancer cells would provide important advantages over current therapies (Juillerat, et al., 2017).

[0003] Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. The ability to engineer synthetic systems in T cells that are responding to multiple inputs would benefit adoptive immunotherapy using engineered T cells (Juillerat, et al., 2017; Chakravarti D. & Wong W. W., 2015). In the last decade numerous approaches to spatiotemporally control engineered T cells, including those relying on the addition of exogenous small molecules or monoclonal antibodies to regulate (Juillerat A, et al., 2016; Wu CY, et al; Ma JS, et al., 2016; Rodgers DT, et al., 2016; Tamada, et al., 2012; Urbanska, et al., 2012) or terminate (Marin V, et al., 2012; Poriot L, et al; Straathof KC., 2005; Duong CP, et al., 2011) engineered T cell functions. Alternatively, to achieve optimal tuning of CAR T cell targeting and functional properties, researchers have developed novel approaches based on the use of combinatorial antigen targeting, such as trans -signaling CARs (Straathof KC., 2005; Duong CP, et al., 2011). Integration of endogenous environmental signals, in addition to antigen recognition, may represent a valuable advancement to improve the control of the CAR T cell technology. An attractive strategy to discriminate between healthy tissue and cancer cells would be to harness the idiosyncrasies of the tumor microenvironment (TME). The tumor microenvironment is characterized by numerous factors including expression of matrix metalloproteinases, cathepsins, proteases, Indolomine 2,3-dioxygenase, low extracellular pH (acidosis) and low oxygenation (hypoxia) (Brown & Wilson, 2004; Vaupel & Mayer, 2007).

[0004] Disclosed herein are approaches that take advantage of the TME to trigger the activation of engineered T cells.

SUMMARY

[0005] Disclosed herein are methods and compositions for inducible T cell therapeutics that take advantage of the tumor microenvironment (TME) to trigger the activation of engineered T cells. Using exogenous factors or known environmental signals within the TME allows the manipulation of the engineered T cell response. In particular, engineered T cells that can only be activated in the presence of certain TME factors will only be activated in the tumor atmosphere, thereby reducing the potential for on-target off-tumor toxicities, but also potentially enhancing the activity of engineered T cells.

[0006] According to one aspect of the present disclosure, provided herein is an recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising (a) a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon; (b) an antibody domain comprising a TAA binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease -cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.

[0007] According to one aspect of the present disclosure, provided herein is an recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising (a) a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 gamma; and (b) an antibody domain comprising a TAA binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.

[0008] According to one aspect of the present disclosure, provided herein is an recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising (a) a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 delta; and (b) an antibody domain comprising an antigen binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.

[0009] According to one aspect of the present disclosure, provided herein is an recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising (a) a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of TCR alpha; and (b) an antibody domain comprising an antigen binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.

[0010] According to one aspect of the present disclosure, provided herein is an recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising (a) a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of TCR beta; and (b) an antibody domain comprising an antigen binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell. In some embodiments, the antibody domain is a human or humanized antibody. In some embodiments, the encoded antigen binding domain is connected to the TCR extracellular domain by a linker sequence. In some embodiments, the encoded linker sequence comprises (G ₄ S) _n , wherein n=l to 4. In some embodiments, the encoded antigen binding domain is connected to the blocking domain by a linker sequence that encodes a protease-cleavable linker. In some embodiments, the linker sequence encodes a matrix metalloproteinase cleavable linker. In some embodiments, the matrix metalloproteinase cleavage linker comprises a sequence selected from any of sequences in Tables 1-14. In some embodiments, the linker sequence encodes a cathepsin-cleavable linker. In some embodiments, the linker sequence encodes a Urokinase plasminogen activator (uPA)-cleavable linker. In some embodiments, the encoded antigen binding domain is connected to an antibody that specifically binds a blocking domain by a linker sequence that encodes a protease-cleavable linker. In some embodiments, the TCR subunit comprises a TCR extracellular domain. In some embodiments, the TCR subunit comprises a TCR transmembrane domain. In some embodiments, the TCR subunit comprises a TCR intracellular domain. In some embodiments, the TCR subunit comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit. In some embodiments, the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one modification thereto. In some embodiments, the TCR subunit comprises an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto. In some embodiments, the antibody domain comprises an antibody fragment. In some embodiments, the antibody domain comprises a scFv or a VHH domain. In some embodiments, the recombinant nucleic acid encodes (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 of an anti-TAA light chain binding domain amino acid sequence with 70-100% sequence identity to a light chain (LC) CDR1, LC CDR2 and LC CDR3 of an anti-TAA light chain binding domain provided herein, respectively, and/or (ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of an anti-TAA heavy chain binding domain amino acid sequence with 70-100% sequence identity to a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of an anti-TAA heavy chain binding domain provided herein, respectively. In some embodiments, the recombinant nucleic acid encodes a light chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a light chain variable region amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity to a light chain variable region amino acid sequence of a light chain variable region provided herein. In some embodiments, the recombinant nucleic acid encodes a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a heavy chain variable region amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to a heavy chain variable region amino acid sequence of a heavy chain variable region provided herein. In some embodiments, the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137,

CD 154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the recombinant nucleic acid molecule further comprises a sequence encoding a costimulatory domain. In some embodiments, the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of DAP 10,

DAP 12, CD30, LIGHT, 0X40, CD2, CD27, CD28, CDS, ICAM-l, LFA-l (CD 11 a/CD 18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto. In some embodiments, the linker sequence encodes a peptide sequence that is cleaved by at least one of a tumor cell surface protease, a carboxypeptidase, a cathepsin, a kallikrein, a hexokinase, a plasmin, a stromelysin, factor Xa, a chymotrypsin-like protease, a trypsin -like protease, a elastase-like protease, atryptase, a chymase, a subtilisin-like protease, an actinidain, a proteinase, a bromelain, a calpain, a caspase, a cysteine protease, a papain, an HIV-1 protease, an HSV protease, a CMV protease, a chymosin, a renin, a pepsin, a matriptase, a plasmepsin, a nepenthesin, a

metalloexopeptidase, a metalloendopeptidase, a matrix metalloproteinase/ collagenase, a plasminogen activator, a urokinase plasminogen activator (uPA), an enterokinase, a prostate-specific antigen (PSA, hK3), an interleukin- 1 b converting enzyme, a thrombin, a fibroblast activation protein (FAP), a meprin, a granzyme, and a dipeptidyl peptidase. In some embodiments, the cathepsin is cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, or cathepsin L; the hexokinase is hKl, hKlO, or hKl5; the proteinase is PR-3; the caspase is caspase-3; the cysteine protease is Mir 1 -CP or legumain; the matrix metalloproteinase or collagenase is MMPl/( interstitial collagenase), MMP2/type IV collagenase,

MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM 10, or ADAM 12; the prostate-specific antigen is PSA or hK3, the FAP is FAP -a; the granzyme is granzyme M or granzyme B; or the dipeptidyl peptidase is dipeptidyl peptidase IV (DPPIV/CD26). In some embodiments, the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP. In some embodiments, the isolated nucleic acid molecule is mRNA. In some embodiments, the TFP includes an immunoreceptor tyrosine-based activation motif (ITAM) of a TCR subunit that comprises an ITAM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CDl6a, CDl6b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto. In some embodiments, the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon. In some embodiments, the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit. In some embodiments, the nucleic acid comprises a nucleotide analog. In some embodiments, the nucleotide analog is selected from the group consisting of 2 -O- methyl, 2' -O-methoxyethyl (2' -O-MOE), 2' -O-aminopropyl, 2' -deoxy, T-deoxy-2' -fluoro, 2' -O- aminopropyl (2' -O-AP), 2'-0-dimethylaminoethyl (2' -O-DMAOE), 2' -O-dimethylaminopropyl (2' -O- DMAP), T-O-dimethylaminoethyloxyethyl (2' -O-DMAEOE), 2 -O-N-methylacetamido (2' -O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1 ' ,5 ' - anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2 -fluoro N3-P5 ' -phosphoramidite. In some embodiments, the recombinant nucleic acid molecule further comprises a leader sequence.

[0011] According to another aspect of the present disclosure, provided herein is a polypeptide molecule encoded by the nucleic acid molecule.

[0012] According to another aspect of the present disclosure, provided herein is a recombinant TFP molecule comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain.

[0013] According to another aspect of the present disclosure, provided herein is a recombinant TFP molecule comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.

[0014] According to another aspect of the present disclosure, provided herein is a recombinant TFP molecule comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally integrating into an endogenous TCR complex. In some embodiments, the TFP molecule further comprises an antibody or antibody fragment comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the anti-TAA binding domain is a scFv or a VH domain. In some embodiments, the anti-TAA binding domain comprises a heavy chain with 95-100% identity to an amino acid sequence of an anti-TAA light chain provided herein, a functional fragment thereof, or an amino acid sequence thereof having at least one but not more than 30 modifications. In some embodiments, the anti-TAA binding domain comprises a light chain with 95-100% identity to an amino acid sequence of an anti-TAA heavy chain provided herein, a functional fragment thereof, or an amino acid sequence thereof having at least one but not more than 30 modifications. In some embodiments, the recombinant TFP molecule comprises a TCR extracellular domain that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma. In some embodiments, the anti-TAA binding domain is connected to the TCR extracellular domain by a linker sequence. In some embodiments, the linker region comprises (G ₄ S) _n , wherein n=l to 4. In some embodiments, the recombinant TFP molecule further comprises a sequence encoding a costimulatory domain. In some embodiments, the recombinant TFP molecule further comprises a sequence encoding an intracellular signaling domain. In some embodiments, the recombinant TFP molecule further comprises a leader sequence.

[0015] According to another aspect of the present disclosure, provided herein is a nucleic acid comprising a sequence encoding a TFP. In some embodiments, the nucleic acid is selected from the group consisting of a DNA and a RNA. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid comprises a nucleotide analog. In some embodiments, the nucleotide analog is selected from the group consisting of 2' -O-methyl, V -O-methoxyethyl (2' -O-MOE), V -O- aminopropyl, 2' -deoxy, T-deoxy-2' -fluoro, V -O-aminopropyl (2' -O-AP), 2'-0-dimethylaminoethyl (2' -O-DMAOE), 2' -O-dimethylaminopropyl (2' -O-DMAP), T-O-dimethylaminoethyloxyethyl (2' -O- DMAEOE), 2' -O-N-methylacetamido (2' -O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1 ' ,5 ' - anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2 ' -fluoro N3-P5 ' - phosphoramidite. In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is an in vitro transcribed nucleic acid. In some embodiments, the nucleic acid further comprises a sequence encoding a poly(A) tail. In some embodiments, the nucleic acid further comprises a 3 ' UTR sequence.

[0016] According to another aspect of the present disclosure, provided herein is a vector comprising a nucleic acid molecule encoding a TFP. In some embodiments, the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector. In some embodiments, the vector further comprises a promoter. In some embodiments, the vector is an in vitro transcribed vector. In some embodiments, a nucleic acid sequence in the vector further comprises a poly(A) tail. In some embodiments, a nucleic acid sequence in the vector further comprises a 3 ' UTR.

[0017] Also provided herein is a cell comprising the recombinant nucleic acid molecule described herein, the polypeptide molecule described herein, the TFP molecule described herein, and/or the vector described herein. In some embodiments, the cell is a human T cell. In some embodiments, the T cell is a CD8+, CD4+ or CD4+CD8+ T cell. In some embodiments, the cell further comprises a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain. In some embodiments, the inhibitory molecule comprise first polypeptide that comprises at least a portion of PD 1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.

[0018] According to another aspect of the present disclosure, provided herein is a human CD8+, CD4+ or CD4+CD8+ T cell comprising at least two TFP molecules, the TFP molecules comprising an anti- TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+, CD4+ or CD4+CD8+ T cell.

[0019] According to another aspect of the present disclosure, provided herein is a protein complex comprising: a TFP molecule comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and at least one endogenous TCR subunit or endogenous TCR complex. In some embodiments, the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, and a CD3 delta TCR subunit. In some embodiments, the anti-TAA binding domain is connected to the TCR extracellular domain by a linker sequence. In some embodiments, the linker region comprises (G ₄ S) _n , wherein n=l to 4.

[0020] According to another aspect of the present disclosure, provided herein is a protein complex comprising a TFP encoded by the recombinant nucleic acid molecule described herein, and at least one endogenous TCR subunit or endogenous TCR complex.

[0021] According to another aspect of the present disclosure, provided herein is a human CD8+, CD4+ or CD4+CD8+ T cell comprising at least two different TFP proteins per the protein complex.

[0022] According to another aspect of the present disclosure, provided herein is a human CD8+, CD4+ or CD4+CD8+ T cell comprising at least two different TFP molecules encoded by the isolated nucleic acid molecule described herein.

[0023] According to another aspect of the present disclosure, provided herein is a population of human CD8+, CD4+ or CD4+CD8+ T cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules, the TFP molecules comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+, CD4+ T or CD4+CD8+ cell.

[0024] According to another aspect of the present disclosure, provided herein is a population of human CD8+, CD4+ or CD4+CD8+ T cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules encoded by the isolated nucleic acid molecule.

[0025] According to another aspect of the present disclosure, provided herein is a method of making a cell comprising transducing a T cell with the recombinant nucleic acid molecule described herein or the vector.

[0026] According to another aspect of the present disclosure, provided herein is a method of generating a population of RNA-engineered cells comprising introducing an in vitro transcribed RNA, a circular RNA or synthetic RNA into a cell, where the RNA comprises a nucleic acid encoding the TFP molecule.

[0027] According to another aspect of the present disclosure, provided herein is a method of providing an anti-tumor immunity in a mammal comprising administering to the mammal an effective amount of the recombinant nucleic acid molecule described herein, the polypeptide molecule described herein, a cell expressing the polypeptide molecule, the TFP molecule, the vector, or the cell. In some

embodiments, the cell is an autologous T cell. In some embodiments, the cell is an allogeneic T cell. In some embodiments, the mammal is a human.

[0028] According to another aspect of the present disclosure, provided herein is a method of treating a mammal having a disease associated with expression of mesothelin comprising administering to the mammal an effective amount of the isolated nucleic acid molecule, the polypeptide molecule, a cell expressing the polypeptide molecule, the TFP molecule, the vector, or the cell. In some embodiments, the disease associated with mesothelin expression is selected from the group consisting of a proliferative disease, a cancer, a malignancy, and a non-cancer related indication associated with expression of mesothelin. In some embodiments, the disease is a cancer selected from the group consisting of mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, thyroid cancer, bladder cancer, ureter cancer, kidney cancer, endometrial cancer, esophogeal cancer, gastric cancer, thymic carcinoma, cholangiocarcinoma and stomach cancer. In some embodiments, the disease is a cancer selected from the group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, endometroid mucinous ovarian carcinoma, pancreatic adenocarcinoma, ductal pancreatic adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, breast adenocarcinoma, a disease associated with mesothelin expression, and combinations thereof. In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that increases the efficacy of a cell expressing a TFP molecule. In some embodiments, less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing an anti-TAA chimeric antigen receptor (CAR). In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule. In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that treats the disease associated with mesothelin.

[0029] Also provided herein is the recombinant nucleic acid molecule, the isolated polypeptide molecule, a cell expressing the polypeptide molecule, the isolated TFP, the vector, the complex, or the cell, for use as a medicament.

[0030] According to another aspect of the present disclosure, provided herein is a method of treating a mammal having a disease associated with expression of mesothelin comprising administering to the mammal an effective amount of the isolated nucleic acid molecule described herein, the polypeptide molecule described herein, a cell expressing the polypeptide molecule described herein, the TFP molecule described herein, the vector described herein, or the cell described herein, wherein less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing an anti-TAA chimeric antigen receptor (CAR).

[0031] According to another aspect, provided herein is a method of treating a mammal having a disease associated with expression of TAA comprising administering to the mammal an effective amount of the isolated nucleic acid molecule described herein, the polypeptide molecule described herein, a cell expressing the polypeptide molecule described herein, the TFP molecule described herein, the nucleic acid described herein, the vector described herein, or the cell described herein.

[0032] In some embodiments, the disease associated with TAA expression is selected from the group consisting of a proliferative disease, a cancer, a malignancy, and a non-cancer related indication associated with expression of TAA. In some embodiments, the disease is a cancer selected from the group consisting of mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, thyroid cancer, bladder cancer, ureter cancer, kidney cancer, endometrial cancer, esophageal cancer, gastric cancer, thymic carcinoma, cholangiocarcinoma and stomach cancer. In some embodiments, the disease is a cancer selected from the group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, endometroid mucinous ovarian carcinoma, pancreatic adenocarcinoma, ductal pancreatic

adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, breast

adenocarcinoma, a disease associated with TAA expression, and combinations thereof. In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that increases the efficacy of a cell expressing a TFP molecule. In some embodiments, less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing an anti-TAA chimeric antigen receptor (CAR). In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule. In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that treats the disease associated with the antigen. In some embodiments, the antigen is a tumor-associated antigen. In some embodiments, the antigen is one or more selected from CD 19, B-cell maturation antigen (BCMA), mesothelin (MSLN), ILl3Rcx2, MUC16, CD22, PD-l, BAFF or BAFF receptor, and ROR-l.

[0033] Also provided herein is the isolated nucleic acid molecule described herein, the isolated polypeptide molecule described herein, a cell expressing the polypeptide molecule described herein, the isolated TFP described herein, the nucleic acid described herein, the vector described herein, the complex described herein, or the cell described herein, for use as a medicament.

[0034] According to another aspect, provided herein is a method of treating a mammal having a disease associated with expression of TAA comprising administering to the mammal an effective amount of the isolated nucleic acid molecule described herein, the polypeptide molecule described herein, a cell expressing the polypeptide molecule described herein, the TFP molecule described herein, the nucleic acid described herein, the vector described, or the cell described herein, wherein less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing an anti -TAA chimeric antigen receptor (CAR).

[0035] In some embodiments, the TFP comprises an anti-TAA binding domain binds to an antigen derived from antigens of alpha-actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDK12, CDKN2A, CLPP, COA-l, CSNK1A1, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD, FNDC3B, FN1, GAS7, GPNMB, HAUS3, HSDL1, LDLR-fucosyltransferase AS fusion protein, HLA-A2d, HLA-A1 ld, hsp70- 2, MART2, MATN, ME1, MUM-lf, MUM-2, MUM-3, neo-PAP, Myosin class I, NFYC, OGT, OS-9, p53, pml-RARalpha fusion protein, PPP1R3B, PRDX5, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, TGF-betaRII, triosephosphate isomerase, BAGE-l, D393-CD20n, Cyclin-Al, GAGE-l, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GnTVf, HERV-K-MEL, KK-LC-l, KM-HN-l, LAGE-l, LY6K, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12 m, MAGE-C1, MAGE-C2, mucink, NA88-A, NY-ESO-l / LAGE-2, SAGE, Spl7, SSX-2, SSX-4, TAG-l, TAG-2, TRAG-3, TRP2- INT2g, XAGE- 1 b/GAGED2a, B7H4, DLL3, TROP-2, Nectin-4, tissue factor, LIV-l, CD48, cMET„ Gene / protein, CEA, gplOO / Pmell7, mammaglobin-A, Melan-A / MART-l, NY-BR-l, OA1, PAP, PSA, RAB38 / NY-MEL-l, TRP-l / gp75, TRP-2, tyrosinase, adipophilin, AIM-2, ALDH1A1, BCLX (L), BING-4, CALCA, CD45, CD274, CPSF, cyclin Dl, DKK1, ENAH (hMena), EpCAM, EphA3, EZH2, FGF5, glypican-3, HER-2/neu, HLA-DOB, Hepsin, IDOl, IGF2B3, ILl3Ralpha2, Intestinal carboxyl esterase, alpha-foetoprotein, Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP, mdm-2, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, p53, PAX5, PBF, PRAME, PSMA, RAGE-l, RGS5, RhoC, RNF43, RU2AS, secemin 1, SOX10, STEAP1, survivin, Telomerase, TPBG, VEGF, and WT1.

In some embodiments, the TFP comprises one or more of an anti-CD 19 binding domain, an anti -B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti- IL 13Ra2 binding domain, an anti-MUCl6 binding domain, an anti-CD22 binding domain, an anti -PD- 1 binding domain, an anti-BAFF or BAFF receptor binding domain, and an anti-ROR-l binding domain.

INCORPORATION BY REFERENCE

[0036] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIGs. 1A-B show two drawings comprising an exemplary CAR T cell bound to HSA via HSA- binding sdAb and a protease-cleavable linker. FIG. 1A shows a CAR T cell fused with an anti -HSA antibody; FIG. IB shows the CAR T cell from FIG. 1A with HSA bound to the anti-HSA domain.

[0038] FIGs. 2A-B show two drawings depicting the fusion of HSA -binding sdAb to the Va or Vb Domain of a TFP T cell via a protease-cleavable linker. FIG. 2A shows a TFP T cell fused with an anti- HSA antibody; FIG. 2B shows the TFP T cell from FIG. 2A with HSA bound to the anti-HSA domain.

[0039] FIGs. 3A-B is two drawings depicting HSA binding to the sdAb of the TFP T cell. This blocks binding of the TCR to the MHC/peptide antigen complex. FIG. 3A shows a TFP T cell fused with an anti-HSA antibody; FIG. 3B shows the TFP T cell from FIG. 3A with HSA bound to the anti-HSA domain.

[0040] FIG. 4 is a drawing depicting proteolytic cleavage of the HSA-binding sdAb at the protease - cleavage site, which enables the inducible TFP T cell to bind to its MHC/peptide complex.

[0041] FIG. 5 is a drawing depicting direct fusion of HSA to an anti -tumor-associated antigen binding domain of a TFP T cell.

[0042] FIG. 6 shows sequences of example anti-HSA-[cleavable-linker]-anti-MSLN constructs. FIG. 6A shows a uPA-cleavable fusion protein sequence. FIG. 6B shows an MMP9vl-cleavable fusion protein sequence. FIG. 6C shows an MMP9v2-cleavable fusion protein sequence. FIG. 6D shows a cathepsin B fusion protein sequence. FIG. 6E shows a non-cleavable control sequence.

[0043] FIG. 7 depicts two graphs showing data demonstrating that inducible anti-mesothelin (MSLN) TFP T cells bind to soluble MSLN and are inhibited by their HSA blocking domains. FIG. 7A depicts a graph showing FACS data that illustrates the anti-HSA/anti-MSLN TFPs are able to bind to MSLN in the absence of HSA, and the constructs are able to bind HSA in the absence of MSLN. FIG. 7B depicts two graphs showing the ability of anti-HSA/anti-MSLN TFP T cells to induce cytokine production when expressed in Jurkat cells and co-cultured with MSTO cells that express MSLN. Panels show from left to right Jurkat cells alone, the anti-HSA/anti-MSLN TFP T cells alone, anti-HSA/anti-MSLN TFP cells comprising an MMP9-cleavable linker, anti-HSA/anti-MSLN TFP T cells comprising a cathepsin B- cleavable linker, and anti-HSA/anti-MSLN TFP cells comprising a non-cleavable linker, and MSTO cells alone (no effector cells, i.e., Jurkat). Each was done in the presence or absence of HSA.

[0044] FIG. 8 is a western blot illustrating cleavability of anti-HSA -cleavable-linker-anti-MSLN ("SD1") constructs. Constructs were loaded pairwise as follows: ctHSA-sdAb-uPA-SDl sdAb digested with uPA +/- FBS; aAlb-N/C-SDl digested with uPA +/- FBS; aHSA sdAb-MMP9vl- SDlsdAb digested with MMP9 +/- FBS; aHSA sdAb-MMP9v2-SDlsdAb digested with MMP9 +/- FBS; and aHSA sdAb-cathepsin B-SD1 sdAb digested with cathepsin B +/- FBS. The western blot was probed with an anti-6His antibody and shows both the uncleaved constructs (around 30kDa) and the cleaved products (around 14 kDa).

DETAILED DESCRIPTION

[0045] Provided herein are compositions of matter and methods of use for the treatment of a disease such as cancer, using inducible T cell receptor (TCR) fusion proteins or T cell populations. As used herein, an "inducible T cell receptor (TCR) fusion protein" includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that, when induced, is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T cell e.g., once the T cell has entered the tumor microenvironment (TME) of a solid tumor. In particular, the inducible TFP T cells disclosed herein comprise a blocking domain that keeps the antigen binding domain (e.g., a tumor associated antigen binding domain) from binding until the TFP T cells reach the target site (e.g., a tumor). Thus, the compositions and methods disclosed herein are suitable for treating a disease wherein the target of interest is expressed on cells of a tissue other than the target tissue, as the inducible nature of the TFP T cells reduces the likelihood of off-target effects in a subject (e.g., a mammal).

[0046] As provided herein, TFPs provide substantial benefits as compared to Chimeric Antigen Receptors. The term "Chimeric Antigen Receptor," or alternatively, a "CAR," refers to a recombinant polypeptide comprising an extracellular antigen binding domain in the form of a binding domain such as an scFv, sdAb, Fab, etc., a transmembrane domain, and cytoplasmic signaling domains (also referred to herein as "an intracellular signaling domains") comprising a functional signaling domain derived from a stimulatory molecule as defined below. Generally, the central intracellular signaling domain of a CAR is derived from the CD3 zeta chain that is normally found associated with the TCR complex. The CD3 zeta signaling domain can be fused with one or more functional signaling domains derived from at least one co-stimulatory molecule such as CD2, 4-1BB (i.e., CD137), CD27 and/or CD28.

[0047] In one aspect, described herein are isolated nucleic acid molecules encoding an inducible T cell Receptor (TCR) fusion protein that comprise a TCR subunit, a non-human, human or humanized antibody domain comprising an anti-tumor associated antigen (TAA) binding domain, and a blocking domain connected to the TAA via a protease-cleavable linker, e.g., a matrix metalloproteinase cleavable linker. In some embodiments, the blocking domain is an antibody such as anti-albumin. In some embodiments, the blocking domain is albumin, IgG, or other bulky protein that sterically hinders binding of the TAA to its target protein on the tumor cell. In some embodiments, the inducible TFP comprises more than one blocking domain and more than one protease-cleavable linker.

[0048] In some embodiments, the TCR subunit comprises a TCR extracellular domain. In other embodiments, the TCR subunit comprises a TCR transmembrane domain. In yet other embodiments, the TCR subunit comprises a TCR intracellular domain. In further embodiments, the TCR subunit comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit. In yet further embodiments, the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one, two or three modifications thereto. In yet further embodiments, the TCR subunit comprises an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of CD2 and/or 4-1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one, two or three modifications thereto.

[0049] In some embodiments, the antibody domain comprises an antibody fragment. In some embodiments, the antibody domain comprises a scFv, a sdAb or a VH domain.

[0050] In some embodiments, the isolated nucleic acid molecules comprise (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 of any anti-tumor-associated antigen light chain binding domain amino acid sequence provided herein, and/or (ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of any anti- tumor-associated antigen heavy chain binding domain amino acid sequence provided herein.

[0051] In some embodiments, the light chain variable region comprises an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein. In other embodiments, the heavy chain variable region comprises an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein.

[0052] In some embodiments, the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma, or a functional fragment thereof, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto. In other embodiments, the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta chain of the TCR or TCR subunits CD3 epsilon, CD3 gamma and CD3 delta, or a functional fragment thereof, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto.

[0053] In some embodiments, the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, functional fragment(s) thereof, and amino acid sequences thereof having at least one, two or three modifications but not more than 20 modifications thereto.

[0054] In some instances, the isolated nucleic acid molecule further comprises a sequence encoding a costimulatory domain. In some instances, the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of DAP 10, DAP 12, CD30, LIGHT, 0X40, CD2, CD27, CD28, CDS, ICAM-l, LFA-l (CD 11 a/CD 18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto. In some instances, the isolated nucleic acid molecule further comprises a leader sequence. In some instances, the isolated nucleic acid molecule is mRNA.

[0055] In some instances, the TFP includes an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP 12), CD5, CDl6a, CDl6b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto. In some instances, the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon. In some instances, the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.

[0056] In some instances, the nucleic acid comprises a nucleotide analog. In some instances, the nucleotide analog is selected from the group consisting of 2' -O-methyl, V -O-methoxyethyl (2' -O- MOE), 2' -O-aminopropyl, V -deoxy, T-deoxy-2' -fluoro, 2' -O-aminopropyl (2' -O-AP), 2'-0- dimethylaminoethyl (2' -O-DMAOE), 2' -O-dimethylaminopropyl (2' -O-DMAP), T-O- dimethylaminoethyloxyethyl (2' -O-DMAEOE), 2' -O-N-methylacetamido (2' -O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1 ' .5 ' - anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2 ' -fluoro N3-P5 ' -phosphoramidite.

[0057] Also provided herein are isolated polypeptide molecules encoded by any of the previously described nucleic acid molecules.

[0058] Also provided herein in another aspect, are isolated T cell receptor fusion protein (TFP) molecules that comprise an anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the isolated TFP molecules comprises an antibody or antibody fragment comprising an anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain.

[0059] In some embodiments, the anti-TAA binding domain is an antibody, Fab, scFv, a VH domain, or an sdAb, or a functional fragment thereof. In other embodiments, the anti-tumor-associated antigen binding domain comprises a light chain and a heavy chain of an amino acid sequence provided herein, or a functional fragment thereof, or an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity with an amino acid sequence provided herein. In some embodiments, the isolated TFP molecules comprise a TCR extracellular domain that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto.

[0060] In some embodiments, the anti-tumor-associated antigen binding domain is connected to the TCR extracellular domain by a non-protease-cleavable linker sequence. In some instances, the linker sequence comprises (G ₄ S) _n , wherein n=l to 4. In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G ₄ S)n, wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G ₄ S) _n , wherein n=l to 3.

[0061] In some embodiments, the isolated TFP molecules further comprise a sequence encoding a costimulatory domain. In other embodiments, the isolated TFP molecules further comprise a sequence encoding an intracellular signaling domain. In yet other embodiments, the isolated TFP molecules further comprise a leader sequence.

[0062] Also provided herein are vectors that comprise a nucleic acid molecule encoding any of the previously described TFP molecules. In some embodiments, the vector is selected from the group consisting of a DNA, an RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector. In some embodiments, the vector further comprises a promoter. In some embodiments, the vector is an in vitro transcribed vector. In some embodiments, a nucleic acid sequence in the vector further comprises a poly(A) tail. In some embodiments, a nucleic acid sequence in the vector further comprises a 3 ' UTR.

[0063] Also provided herein are cells that comprise any of the described vectors. In some embodiments, the cell is a human T cell. In some embodiments, the cell is a CD8+ or CD4+ or CD4+CD8+ T cell. In one embodiment, the CD8+ cell is a gamma-delta T cell. In another embodiment, the CD8+ cell is an NK-T cell. In other embodiments, the cells further comprise a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain. In some instances, the inhibitory molecule comprises a first polypeptide that comprises at least a portion of PD1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.

[0064] In another aspect, provided herein are isolated TFP molecules that comprise a human or humanized anti-tumor-associated antigen (TAA) binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.

[0065] In another aspect, provided herein are isolated TFP molecules that comprise an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally integrating into an endogenous TCR complex.

[0066] In another aspect, provided herein are human CD8+ or CD4+ or CD4+CD8+ T cells that comprise one or more inducible TFP molecules, the TFP molecules comprising a blocking domain or antibody specific to a protein suitable for a blocking domain, an antigen binding domain (e.g., an anti- tumor-associated (TAA) antigen binding domain), a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+ or CD4+ or CD4+CD8+ T cell. In another aspect, the cells comprise at least two non-identical inducible TFP molecules.

[0067] In another aspect, provided herein are protein complexes that comprise i) a TFP molecule comprising an anti-tumor-associated antigen binding domain, a blocking domain or an antibody specific to a blocking domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and ii) at least one endogenous TCR complex subunit.

[0068] In some embodiments, the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma. In some embodiments, the anti-tumor-associated antigen binding domain is connected to the TCR extracellular domain by a linker sequence. In some instances, the linker region comprises (G ₄ S) _n , wherein n=l to 4. In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G ₄ S) _n , wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G ₄ S) _n , wherein n=l to 3.

[0069] Also provided herein are human CD8+ or CD4+ or CD8+CD4+ T cells that comprise at least two different TFP molecules per any of the described protein complexes.

[0070] In another aspect, provided herein is a population of human CD8+ or CD4+ or CD8+CD4+ or NK T cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules, the TFP molecules comprising an antigen binding domain (e.g., an anti-tumor-associated antigen binding domain), a blocking domain or an antibody to a protein suitable for a blocking domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+ or CD4+ or CD4+CD8+ T cell.

[0071] In another aspect, provided herein is a population of human CD8+ or CD4+ T or CD8+CD4+ cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules encoded by an isolated nucleic acid molecule provided herein.

[0072] In another aspect, provided herein are methods of making a cell comprising transducing a T cell with any of the described vectors.

[0073] In another aspect, provided herein are methods of generating a population of RNA-engineered cells that comprise introducing an in vitro transcribed RNA or synthetic RNA into a cell, where the RNA comprises nucleic acid encoding one or more of the described TFP molecules. In some embodiments the RNA is a circular RNA. [0074] In another aspect, provided herein are methods of providing an anti-tumor immunity in a mammal that comprise administering to the mammal an effective amount of a cell expressing any of the described TFP molecules. In some embodiments, the cell is an autologous T cell. In some embodiments, the cell is an allogeneic T cell. In some embodiments, the mammal is a human.

[0075] In another aspect, provided herein are methods of treating a mammal having a disease associated with expression of tumor-associated antigen that comprise administering to the mammal an effective amount of the cell comprising any of the described TFP molecules. In some embodiments, the disease associated with tumor-associated antigen expression is selected from a proliferative disease such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia, or is a non-cancer related indication associated with expression of tumor-associated antigen.

[0076] In some embodiments, the disease is a hematologic cancer selected from the group consisting of one or more acute leukemias including but not limited to B-cell acute lymphoid leukemia ("B-ALL"), T cell acute lymphoid leukemia ("T-ALL"), acute lymphoblastic leukemia (ALL); one or more chronic leukemias including but not limited to chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematologic cancers or hematologic conditions including, but not limited to B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt' s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell- follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, smoldering multiple myeloma, solitary plasmacytoma, lymphoplasmacytic lymphoma, plasma cell leukemia, myelodysplasia and

myelodysplastic syndrome, non-Hodgkin' s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom' s macroglobulinemia, and "preleukemia" which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and to disease associated with tumor-associated antigen expression include, but not limited to atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing tumor- associated antigen; and combinations thereof.

[0077] In some embodiments, the disease is a solid tumor. In some instances, the cancer is selected from the group consisting of renal cell carcinoma, breast cancer, lung cancer, mesothelioma, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, and stomach cancer. In some embodiments the tumor associated antigen is expressed on the surface of tumor cells and is suitable for treatment with a TFP T cell (comprising a T cell receptor Fusion Protein (TFP) T cell as disclosed herein, or a CAR T cell. In some embodiments the tumor associated antigen is expressed inside the cancer cell and a suitable treatment is a TCR T cell.

[0078] In some embodiments, the cells expressing any of the described TFP molecules are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule. In some embodiments, the cells expressing any of the described TFP molecules are administered in combination with an agent that treats the disease associated with tumor- associated antigen.

[0079] Also provided herein are any of the described isolated nucleic acid molecules, any of the described isolated polypeptide molecules, any of the described isolated TFPs, any of the described protein complexes, any of the described vectors or any of the described cells for use as a medicament. Definitions

[0080] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0081] The term "a" and "an" refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0082] As used herein, "about" can mean plus or minus less than 1 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or greater than 30 percent, depending upon the situation and known or knowable by one skilled in the art. The term "about" or "approximately" can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term "about" or "approximately" can mean within an order of magnitude, within 5-fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed. The term "about" can have the meaning as commonly understood by one of ordinary skill in the art. The term "about" can refer to +10%. The term "about" can refer to +5%.

[0083] As used herein the specification, "subject" or "subjects" or "individuals" may include, but are not limited to, mammals such as humans or non-human mammals, e.g., domesticated, agricultural or wild, animals, as well as birds, and aquatic animals. "Patients" are subjects suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.

[0084] As used herein, "treating" or "treatment" refers to any indicia of success in the treatment or amelioration of the disease or condition. Treating can include, for example, reducing, delaying or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient. As used herein, "treat or prevent" is sometimes used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition and

contemplates a range of results directed to that end, including but not restricted to prevention of the condition entirely.

[0085] As used herein, "preventing" refers to the prevention of the disease or condition, e.g., tumor formation, in the patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present invention and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual.

[0086] As used herein, a "therapeutically effective amount" is the amount of a composition or an active component thereof sufficient to provide a beneficial effect or to otherwise reduce a detrimental non- beneficial event to the individual to whom the composition is administered. By "therapeutically effective dose" herein is meant a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g. Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999))

[0087] As used herein, a "T cell receptor (TCR) fusion protein" or "TFP" includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T cell.

[0088] The portion of the TFP composition comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) derived from a murine, humanized or human antibody (Harlow et ah, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et ah, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et ah, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et ah, 1988, Science 242:423-426). In one aspect, the antigen binding domain of a TFP composition comprises an antibody fragment. In a further aspect, the TFP comprises an antibody fragment that comprises a scFv or a sdAb.

[0089] The term "antigen" or "Ag" may refer to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both. As used herein, the term "cancer antigen" or "cancer-related antigen" may refer to any cancer cell marker expressed on the surface of a malignant or tumor cell that can be treated with the combination therapy described herein, including, but not limited to: described herein include, but are not limited to, 5T4, 8H9, o _ί n b Q integrin, anb6 integrin, alphafetoprotein (AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9),

CD 19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171, carcinoembryonic antigen (CEA), EpCAM (epithelial cell adhesion molecule), E-cadherin, EMA

(epithelial membrane antigen), EGFRvlll, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbBl/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3/HER3, ErbB4, epithelial tumor antigen (ETA), folate binding protein (FBP), fetal acetylcholine receptor (AchR), folate rcccptor-a. ganglioside 2 (GD2), ganglioside 3 (GD3), HLA-A1, HLA-A2, high molecular weight melanoma-associated antigen (HMW-MAA), IL-13 receptor «2 ( I L- 13Ra2). kinase insert domain receptor (KDR), k-light chain, Fewis Y (FeY), Fl cell adhesion molecule, melanoma-associated antigen (MAGE-A1 ), mesothelin, mucin-l (MUC1 ), mucin-l6 (MUC16), natural killer group 2 member D (NKG2D) ligands, nerve cell adhesion molecule (NCAM), NY-ESO-l, B7H4, DLL3, TROP-2, Nectin-4, tissue factor, LIV-l, CD48, cMET, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate -specific membrane antigen (PSMA), receptor-tyrosine kinase-like orphan receptor 1 (R0R1), TAA targeted by mAb IgE, tumor- associated glycoprotein-72 (TAG-72), tyrosinase, and vascular endothelial growth factor (VEGF) receptors.

[0090] The term "antibody," as used herein, refers to a protein, or polypeptide sequences derived from an immunoglobulin molecule, which specifically binds to an antigen. Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.

[0091] The terms "antibody fragment" or "antibody binding domain" refer to at least one portion of an antibody, or recombinant variants thereof, that contains the antigen binding domain, i.e., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab' , F(ab' )2, and Fv fragments, single-chain (sc)Fv ("scFv") antibody fragments, linear antibodies, single domain antibodies (abbreviated "sdAb") (either VF or VH), camelid VHH domains, and multi -specific antibodies formed from antibody fragments.

[0092] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

[0093] "Heavy chain variable region" or "VH" (or, in the case of single domain antibodies, e.g., nanobodies, "VHH" or "sdAb") with regard to an antibody refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0094] Unless specified, as used herein an scFv may have the VF and VH regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VF-linker- VH or may comprise VH-linker-VF.

[0095] The portion of the TFP composition of the invention comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb) or heavy chain antibodies HCAb 242:423-426). In one aspect, the antigen binding domain of a TFP composition of the invention comprises an antibody fragment. In a further aspect, the TFP comprises an antibody fragment that comprises a scFv or a sdAb. [0096] The term "antibody heavy chain," refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.

[0097] The term "antibody light chain," refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (" K ") and lambda ("l") light chains refer to the two major antibody light chain isotypes.

[0098] The term "recombinant antibody" refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art. The term "antigen" or "Ag" refers to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both.

[0099] As used herein, the term "combination therapy" means a therapy strategy that embraces the administration of therapeutic compositions of the present invention (e.g., conjugates comprising one or more neoantigens) and one or more additional therapeutic agents as part of a specific treatment regimen intended to provide a beneficial (additive or synergistic) effect from the co-action of these therapeutic agents. Administration of these therapeutic agents in combination may be carried out over a defined time period (usually minutes, hours, days, or weeks depending upon the combination selected). In

combination therapy, combined therapeutic agent may be administered in a sequential manner, or by substantially simultaneous administration.

[0100] As used herein, the terms "cytotoxic T cell (TC)" or "cytotoxic T lymphocyte (CTL)", or "T- killer cells", or "CD8+ T cell" or "killer T cell" are used interchangeably. This type of white blood cells are T lymphocytes that can recognize abnormal cells including cancer cells, cells that are infected particularly by viruses, and cells that are damaged in other ways and induce the death of such cells.

[0101] As used herein, the term "epitope" means a small peptide structure formed by contiguous amino acids, or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and about 9, or about 8-15 amino acids. A T cell epitope means a peptide which can be bound by the MHC molecules of class I or II in the form of a peptide-presenting MHC molecule or MHC complex and then, in this form, be recognized and bound by native T cells, cytotoxic T- lymphocytes or T-helper cells, respectively.

[0102] As used herein, the term "immune cell" refers to a cell that is capable of participating, directly or indirectly, in an immune response. Immune cells include, but are not limited to T cells, B-cells, antigen presenting cells, dendritic cells, natural killer (NK) cells, natural killer T (NK) cells, lymphokine- activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans' cells, stem cells, peripheral blood mononuclear cells, cytotoxic T cells, tumor infiltrating lymphocytes (TIL), etc. An "antigen presenting cell" (APC) is a cell that is capable of activating T cells, and includes, but is not limited to, monocytes/macrophages, B cells and dendritic cells (DCs). "Dendritic cell" or "DC" refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their distinctive morphology, high levels of surface MHC -class II expression. DCs can be isolated from a number of tissue sources. DCs have a high capacity for sensitizing MHC- restricted T cells and are very effective at presenting antigens to T cells in situ. The antigens may be self-antigens that are expressed during T cell development and tolerance, and foreign antigens that are present during normal immune processes. As used herein, an "activated DC" is a DC that has been pulsed with an antigen and capable of activating an immune cell. "T cell" as used herein, is defined as a thymus-derived cell that participates in a variety of cell- mediated immune reactions, including CD8+ T cell and CD4+ T cell.

[0103] As used herein, the term "immune response" means a defensive response a body develops against "foreigner" such as bacteria, viruses and substances that appear foreign and harmful. An anti -cancer immune response refers to an immune surveillance mechanism by which a body recognizes abnormal tumor cells and initiates both the innate and adaptive of the immune system to eliminate dangerous cancer cells.

[0104] The innate immune system is a non-specific immune system that comprises the cells (e.g.,

Natural killer cells, mast cells, eosinophils, basophils; and the phagocytic cells including macrophages, neutrophils, and dendritic cells) and mechanisms that defend the host from infection by other organisms. An innate immune response can initiate the productions of cytokines, and active complement cascade and adaptive immune response. The adaptive immune system is specific immune system that is required and involved in highly specialized systemic cell activation and processes, such as antigen presentation by an antigen presenting cell; antigen specific T cell activation and cytotoxic effect.

[0105] The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.

[0106] "Specific dissociation," as used herein, refers to a separation of two components that preferentially occurs in a particular environment. In an aspect of the invention, specific dissociation refers to dissociation of domains separated by proteolytic cleavage by a protease that is more highly available in the tumor microenvironment. In an aspect of the invention, specific dissociation refers to separation of components of the pharmaceutical composition upon enzymatic digestion or other enzymatic activity.

[0107] As used herein, the phrase "biologically active domain" refers to any protein structure able to modulate a biological activity. This can include, but is not limited to, cytokines, chemokines, antibodies, antibody-drug conjugates, T cell fusion proteins, chimeric antigen receptors (CARs), and T cell receptor subunits.

[0108] The term "anti -tumor effect" refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumor cell proliferation, decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.

[0109] As used herein, the terms "adoptive cellular immunotherapy'" or "adoptive immunotherapy ' or "T cell immunotherapy'", or "Adoptive T cell therapy (ACT)", are used interchangeably. Adoptive immunotherapy uses T cells that a natural or genetically engineered reactivity to a patient's cancer are generated in vitro and then transferred back into the cancer patient. The injection of a large number of activated tumor specific T cells can induce complete and durable regression of cancers.

[0110] The term "autologous" refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0111] The term "allogeneic" refers to any material derived from a different animal of the same species or different patient as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

[0112] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0113] The term "cancer" may refer to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include, but are not limited to, prostate cancer, breast cancer, melanoma, sarcoma, colorectal cancer, pancreatic cancer, uterine cancer, ovarian cancer, stomach cancer, gastric cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, cholangiocarcinoma, squamous cell lung cancer, mesothelioma, adrenocortico carcinoma, esophageal cancer, head & neck cancer, liver cancer, nasopharyngeal carcinoma, neuroepithelial cancer, adenoid cystic carcinoma, thymoma, chronic lymphocytic leukemia, glioma, glioblastoma multiforme, neuroblastoma, papillary renal cell carcinoma, mantle cell lymphoma, lymphoblastic leukemia, acute myeloid leukemia, and the like.

[0114] The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a TFP of the invention can be replaced with other amino acid residues from the same side chain family and the altered TFP can be tested using the functional assays described herein.

[0115] The term "stimulation" refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex.

Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.

[0116] The term "stimulatory molecule" or "stimulatory domain" refers to a molecule or portion thereof expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway. In one aspect, the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or "ITAM". Examples of an ITAM containing primary cytoplasmic signaling sequence that is of particular use in the invention includes, but is not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS") and CD66d.

[0117] The term "antigen presenting cell" or "APC" refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC' s) on its surface. T cells may recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0118] An "intracellular signaling domain," as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the TFP containing cell, e.g., a TFP -expressing T cell. Examples of immune effector function, e.g., in a TFP -expressing T cell, include cytolytic activity and T helper cell activity, including the secretion of cytokines. In an embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In an embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain.

Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.

[0119] A primary intracellular signaling domain can comprise an ITAM ("immunoreceptor tyrosine- based activation motif"). Examples of ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d DAP 10 and DAP 12.

[0120] The term "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Costimulatory molecules include, but are not limited to, an MHC class 1 molecule, BTLA, a Toll ligand receptor,

0X40, CD2, CD27, CD28, CDS, ICAM-l, LFA-l (CDl la/CDl8) and 4-1BB (CD137). A costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. A

costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4- 1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like. The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof. The term "4-1BB" refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Ace. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like; and a "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Ace. No. AAA62478.2, or equivalent residues from non-human species, e.g., mouse, rodent, monkey, ape and the like.

[0121] The term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0122] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence " includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or a RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain one or more introns.

[0123] The term "effective amount" or "therapeutically effective amount" are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological or therapeutic result.

[0124] The term "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

[0125] The term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0126] The term "expression" refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

[0127] The term "transfer vector" refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0128] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0129] The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lenti viruses.

[0130] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic include, but are not limited to, e.g., the Lentivector® gene delivery technology from Oxford BioMedica, the

LentiMax® vector system from Lentigen, and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0131] The term "homologous" or "identity" refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.

[0132] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab' , F(ab' )2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary -determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non human residues. Furthermore, a humanized antibody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. In general, the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al, Nature, 332: 323-329,

1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0133] "Human" or "fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.

[0134] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0135] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0136] The term "operably linked" or "transcriptional control" refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.

[0137] The term "parenteral" administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, intratumoral, or infusion techniques.

[0138] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et ah, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et ah, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et ak, Mol. Cell. Probes 8:91-98 (1994)).

[0139] The terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein' s or peptide' s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0140] The term "promoter" refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0141] The term "promoter/regulatory sequence" refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0142] The term "constitutive" promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0143] The term "inducible" promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

[0144] The term "tissue-specific" promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0145] The terms "linker" and "flexible polypeptide linker" (not to be confused with "protease- cleavable linker" of the TFP molecules disclosed herein) as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly- Ser) _n , where n is a positive integer equal to or greater than 1. For example, n=l, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 and h=10. In one embodiment, the flexible polypeptide linkers include, but are not limited to, (Gly ₄ Ser) ₄ or (Gly ₄ Ser) ₃ . In another embodiment, the linkers include multiple repeats of (Gly ₂ Ser), (GlySer) or (Gly ₃ Ser). Also included within the scope of the invention are linkers described in

WO2012/138475 (incorporated herein by reference). In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G ₄ S)n, wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S)n, wherein n=l to 3.

[0146] As used herein, a 5 ' cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the "front" or 5 ' end of a eukaryotic messenger RNA shortly after the start of transcription. The 5 ' cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co -transcriptionally, such that each influences the other. Shortly after the start of transcription, the 5 ' end of the mRNA being synthesized is bound by a cap -synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.

[0147] As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA, which has been synthesized in vitro. Generally, the in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA. In some embodiments, an in vitro transcribed RNA is circularizable.

[0148] As used herein, a "poly(A)" is a series of adenosines attached by polyadenylation to the mRNA. In the preferred embodiment of a construct for transient expression, the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.

[0149] As used herein, "polyadenylation" refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3 ' end. The 3 ' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3 ' end at the cleavage site.

[0150] As used herein, "transient" refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.

[0151] The term "signal transduction pathway" refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase "cell surface receptor" includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.

[0152] The term "subject" is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human).

[0153] The term, a "substantially purified" cell refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0154] The term "therapeutic" as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.

[0155] The term "prophylaxis" as used herein means the prevention of or protective treatment for a disease or disease state.

[0156] In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to antigens that are common to specific hyperproliferative disorders. In certain aspects, the hyperproliferative disorder antigens of the present invention are derived from, cancers including but not limited to primary or metastatic melanoma, mesothelioma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, NHL, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.

[0157] The term "transfected" or "transformed" or "transduced" refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

The term "specifically binds," refers to an antibody, an antibody fragment or a specific ligand, which recognizes and binds a cognate binding partner (e.g., BCMA, MSLN, NKG2D, ROR1, etc.) present in a sample, but which does not necessarily and substantially recognize or bind other molecules in the sample.

[0158] The term 'binding ligand" may generally refer to a polypeptide (e.g., a protein), a

polynucleotide (e.g., DNA, RNA, or a hybrid of DNA and RNA), a molecule, a chemical compound, a fragment thereof, and/or a hybrid thereof. In some embodiments, the binding ligand can comprise a polynucleotide, and the polynucleotide can be single stranded, double stranded, or a combination thereof. In some embodiments, a binding ligand can comprise a biological molecule or a non -biological molecule. In some embodiments, a biological molecule or non-biological molecule can be a naturally occurring molecule or an artificial molecule. Non-limiting examples of a binding ligand include a protein, a carbohydrate, a lipid, or a nucleic acid. In some embodiments, the binding ligand may associate, bind, and/or couple with an antibody or fragment thereof (e.g., an IgA isotype antibody, an IgD isotype antibody, an IgE isotype antibody, an IgG isotype antibody, an IgM isotype antibody, an IgW isotype antibody, an IgY isotype antibody). In some embodiments the antibody or fragment thereof may be an Fc domain of the antibody (e.g., the binding ligand is an Fc receptor). For example, in some

embodiments the binding ligand can specifically bind to an IgGl antibody. In some embodiments, the binding ligand may be capable of associating, capable of binding, and/or capable of coupling with an antibody or fragment thereof. In some embodiments, the binding ligand can comprise multiple subunits. In some embodiments, a binding ligand can comprise multiple subunits, and the subunits can be the same. In some embodiments, a binding ligand can comprise multiple different subunits. In some embodiments, a binding ligand can comprise multiple subunits, and at least two of the subunits can be different. In some embodiments, a binding ligand can comprise a dimer, trimer, tetramer, pentamer, hexamer, heptamer, nonamer, or decamer. In some embodiments, a binding ligand can comprise greater than about ten subunits. In some embodiments, a binding ligand can comprise a polymer. In some embodiments, the binding ligand may be non-human (e.g., primate), human, or humanized.

[0159] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.

T cell receptor (TCR) fusion proteins (TFP)

[0160] The present invention encompasses recombinant DNA constructs encoding TFPs, wherein the TFP in one aspect comprises an antibody fragment that binds specifically to one or more tumor associated antigens ("TAA"), e.g., a human TAA, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof. In some aspects, the TFPs comprise an intervening in-frame sequence comprising a protease -cleavable linker (e.g., between the sequence encoding the antibody fragment and the sequence encoding the TCR subunit or portion thereof, such that the expressed protein comprises a protease- cleavable site between the TCR subunit and the antibody fragment). The TFPs provided herein are able to associate with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR subunits in order to form a functional TCR complex. In some embodiments, the antigen-binding domain is a humanized or human anti-TAA binding domain. In some embodiments, the antibody fragment is an anti-mesothelin antibody or a fragment thereof. In one aspect, the portion of the TFP comprising the antigen binding domain comprises an antigen binding domain that targets mesothelin. In one aspect, the antigen binding domain targets human mesothelin. Thus, in one aspect, the antigen-binding domain comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the humanized or human anti-mesothelin binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain

complementary determining region 3 (LC CDR3) of a humanized or human anti-mesothelin binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-mesothelin binding domain described herein, e.g., a humanized or human anti-mesothelin binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. In one embodiment, the humanized or human anti-mesothelin binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-mesothelin binding domain described herein, e.g., the humanized or human anti-mesothelin binding domain has two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein. In one embodiment, the humanized or human anti-mesothelin binding domain comprises a humanized or human light chain variable region described herein and/or a humanized or human heavy chain variable region described herein. In one embodiment, the humanized or human anti-mesothelin binding domain comprises a humanized heavy chain variable region described herein, e.g., at least two humanized or human heavy chain variable regions described herein. In one embodiment, the anti-mesothelin binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence provided herein.

[0161] In one embodiment, the humanized or human anti-TAA binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a humanized or human anti-TAA binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-TAA binding domain described herein, e.g., a humanized or human anti-TAA binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. In one embodiment, the humanized or human anti-TAA binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-TAA binding domain described herein, e.g., the humanized or human anti- TAA binding domain has two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein. In one embodiment, the humanized or human anti-TAA binding domain comprises a humanized or human light chain variable region described herein and/or a humanized or human heavy chain variable region described herein. In one embodiment, the humanized or human anti-TAA binding domain comprises a humanized heavy chain variable region described herein, e.g. , at least two humanized or human heavy chain variable regions described herein. In one embodiment, the anti-TAA binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence provided herein. In some embodiments, the TFP comprises an anti-TAA binding domain binds to an antigen derived from antigens of alpha-actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B- RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDK12, CDKN2A, CLPP, COA-l, CSNK1A1, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD,

FNDC3B, FN1, GAS7, GPNMB, HAUS3, HSDL1, LDLR-fucosyltransferase AS fusion protein, HLA- A2d, HLA-A1 ld, hsp70-2, MART2, MATN, ME1, MUM-lf, MUM-2, MUM-3, neo-PAP, Myosin class I, NFYC, OGT, OS-9, p53, pml-RARalpha fusion protein, PPP1R3B, PRDX5, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, TGF-betaRII, triosephosphate isomerase, BAGE-l, D393-CD20n, Cyclin-Al, GAGE-l, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GnTVf, HERV-K-MEL, KK-LC-l, KM-HN-l, LAGE-l, LY6K, MAGE- Al, MAGE-A2, MAGE -A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12 m, MAGE- Cl, MAGE-C2, mucink, NA88-A, NY-ESO-l / LAGE-2, SAGE, Spl7, SSX-2, SSX-4, TAG-l, TAG-2, TRAG-3, TRP2-INT2g, XAGE- lb/GAGED2a, B7H4, DLL3, TROP-2, Nectin-4, tissue factor, LIV-l, CD48, cMET„ Gene / protein, CEA, gplOO / Pmell7, mammaglobin-A, Melan-A / MART-l, NY-BR-l, OA1, PAP, PSA, RAB38 / NY-MEL-l, TRP-l / gp75, TRP-2, tyrosinase, adipophilin, AIM-2,

ALDH1A1, BCLX (L), BING-4, CALCA, CD45, CD274, CPSF, cyclin Dl, DKK1, ENAH (hMena), EpCAM, EphA3, EZH2, FGF5, glypican-3, HER-2/neu, HLA-DOB, Hepsin, IDOl, IGF2B3,

ILl3Ralpha2, Intestinal carboxyl esterase, alpha-foetoprotein, Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP, mdm-2, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, p53, PAX5, PBF, PRAME, PSMA, RAGE-l, RGS5, RhoC, RNF43, RU2AS, secemin 1, SOX10, STEAP1, survivin, Telomerase, TPBG, VEGF, and WT1. In some embodiments, the TFP comprises one or more of an anti-CDl9 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-IL 13Ra2 binding domain, an anti-MUCl6 binding domain, an anti-CD22 binding domain, an anti -PD- 1 binding domain, an anti-BAFF or BAFF receptor binding domain, and an anti-ROR-l binding domain.

Blocking Domains

[0162] The TFP constructs disclosed herein comprise a blocking domain. In some embodiments, the blocking domain is tethered to the antigen binding domain via a protease-cleavable linker. In other embodiments, the blocking domain is tethered to a TCR complex subunit via a protease-cleavable linker. The blocking domain may sterically hinder the binding of the engineered T cell to the target antigen. In an embodiment, the blocking domain is albumin. In an embodiment, the blocking domain is an IgG. In an embodiment, the blocking domain is a peptide capable of sterically blocking the activity of a biologically active molecule, e.g., the binding of the TAA -binding domain to its target. In some embodiments, the TFP does not comprise a blocking domain but rather comprises an antibody specific to a protein suitable for blocking activity of the TFP. In one embodiment, the TFP comprises an anti-HSA antibody.

[0163] Human serum albumin (HSA) (molecular mass ~67 kDa) is the most abundant protein in plasma, present at about 50 mg/ml (600 mM), and has a serum half-life of around 20 days in humans. HSA serves to maintain plasma pH, contributes to colloidal blood pressure, functions as carrier of many metabolites and fatty acids, and serves as a major drug transport protein in plasma. In some embodiments, the serum albumin is human; in some embodiments, the serum albumin is mouse or cynomolgus monkey albumin. In some embodiments, the serum albumin is the same serum albumin as is natively found in the subject to which the albumin-modified protein will be administered (see, e.g., U.S. Patent No. 9,920, 115).

[0164] In one aspect, the TFP of the invention comprises a target -specific binding element otherwise referred to as an antigen binding domain. The choice of moiety depends upon the type and number of target antigen that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize a target antigen that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that may act as target antigens for the antigen binding domain in a TFP of the invention include those associated with viral, bacterial and parasitic infections; autoimmune diseases; and cancerous diseases (e.g., malignant diseases). In some embodiments, the antigen is a tumor-associated antigen.

[0165] In some embodiments, the blocking domain (e.g., albumin or another bulky protein) will be attached covalently directly to the antigen-binding domain (e.g., wherein the antigen-binding domain is attached to the TFP T cell via a protease-cleavable linker). In some embodiments, the albumin will be attached via a short linker (e.g., a protease-cleavable linker). In some embodiments, a subject' s own albumin will be attracted to the antigen-binding domain via an albumin-binding protein, such as an antibody or fragment thereof. In some embodiments, a single albumin molecule, directly attached or attracted via an albumin binding protein, will be enough to block activity. In some embodiments, two or more albumin molecules, directly attached or attracted via an albumin binding protein, will be used.

[0166] In one aspect, the TFP-mediated T cell response can be directed to an antigen of interest by way of engineering an antigen-binding domain into the TFP that specifically binds a desired antigen.

[0167] The antigen binding domain can be any domain that binds to the antigen. In some embodiments, the antigen binding domain comprises an antibody or a fragment thereof, including but not limited to: a monoclonal antibody; a polyclonal antibody; a recombinant antibody; a human antibody; a humanized antibody or a functional fragment thereof, including but not limited to a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of a camelid-derived nanobody; and to an alternative scaffold known in the art to function as antigen binding domain, such as a recombinant fibronectin domain, anticabn, DARPIN and the like. Likewise, a natural or synthetic ligand specifically recognizing and binding the target antigen can be used as antigen binding domain for the TFP, e.g., an NKG2D dimer or another binder comprising, e.g., a binder that participates in a receptor-ligand interaction. In some instances, it is beneficial for the antigen binding domain to be derived from the same species in which the TFP will ultimately be used in. For example, for use in humans, it may be beneficial for the antigen binding domain of the TFP to comprise human or humanized residues for the antigen binding domain of an antibody or antibody fragment.

[0168] Thus, in one aspect, the antigen-binding domain comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the humanized or human anti-TAA binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a humanized or human anti-TAA binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-TAA binding domain described herein, e.g., a humanized or human anti-TAA binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. In one embodiment, the humanized or human anti-TAA binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-TAA binding domain described herein, e.g., the humanized or human anti-tumor-associated antigen binding domain has two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein. In one embodiment, the humanized or human anti-TAA binding domain comprises a single domain (sdAb) antibody. In one embodiment, the humanized or human anti-tumor- associated antigen binding domain comprises a humanized or human light chain variable region described herein and/or a humanized or human heavy chain variable region described herein. In one embodiment, the humanized or human anti-tumor-associated antigen binding domain comprises a humanized heavy chain variable region described herein, e.g., at least two humanized or human heavy chain variable regions described herein. In one embodiment, the anti-tumor-associated antigen binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence provided herein. In an embodiment, the anti -tumor-associated antigen binding domain (e.g., an scFv or V _H H) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity with an amino acid sequence provided herein; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30,

20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein. In one embodiment, the humanized or human anti-tumor-associated antigen binding domain is a scFv, and a light chain variable region comprising an amino acid sequence described herein, is attached to a heavy chain variable region comprising an amino acid sequence described herein, via a linker, e.g., a linker described herein. In one embodiment, the humanized anti-tumor-associated antigen binding domain includes a (Gly ₄ -Ser) _n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4. The light chain variable region and heavy chain variable region of a scFv can be, e.g., in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region. In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comp rises (G ₄ S) _n , wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S)n, wherein n=l to 3.

[0169] In some aspects, a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof. In one aspect, the antigen binding domain is humanized.

[0170] A humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g., European Patent Nos. EP592106 and EP519596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et ah, 1994, Protein Engineering, 7(6):805-8l4; and Roguska et ah, 1994, PNAS, 91:969-973, each of which is incorporated herein by its entirety by reference), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in its entirety by reference), and techniques disclosed in, e.g., U.S. Patent Application Publication No. US20050042664, U.S. Patent Application Publication No. US20050048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. W09317105, Tan et ah, J. Immunol., 169: 1119-25 (2002), Caldas et al., Protein Eng., l3(5):353-60 (2000), Morea et ah, Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(l0):895-904 (1996), Couto et ak, Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al,

J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein in its entirety by reference. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, for example improve, antigen binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.)

[0171] A humanized antibody or antibody fragment has one or more amino acid residues remaining in it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. As provided herein, humanized antibodies or antibody fragments comprise one or more CDRs from nonhuman

immunoglobulin molecules and framework regions wherein the amino acid residues comprising the framework are derived completely or mostly from human germline. Multiple techniques for

humanization of antibodies or antibody fragments are well-known in the art and can essentially be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239,400; PCT Publication No. WO 91/09967; and U.S. Patent Nos. 4,816,567;

6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference in their entirety). In such humanized antibodies and antibody fragments, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. Humanized antibodies are often human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing

(EP592106; EP519596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et ak, Protein Engineering, 7(6):805-8l4 (1994); and Roguska et ak, PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Patent No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

[0172] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable -domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et ak, J. Immunol., 151:2296 (1993); Chothia et ak, J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (see, e.g., Nicholson et ak Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et ak, Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et ak, J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference herein in their entirety). In some embodiments, the framework region, e.g., all four framework regions, of the heavy chain variable region are derived from a VH4-4-59 germline sequence. In one embodiment, the framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence. In one embodiment, the framework region, e.g., all four framework regions of the light chain variable region are derived from a VK3-1.25 germline sequence. In one embodiment, the framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence.

[0173] In some aspects, the portion of a TFP composition of the invention that comprises an antibody fragment is humanized with retention of high affinity for the target antigen and other favorable biological properties. According to one aspect of the invention, humanized antibodies and antibody fragments are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody or antibody fragment characteristic, such as increased affinity for the target antigen, is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0174] In one aspect, the anti-tumor-associated antigen binding domain is a fragment, e.g., a single chain variable fragment (scFv) or a camelid heavy chain (VHH). In one aspect, the anti-tumor-associated antigen binding domain is a Fv, a Fab, a (Fab' )2, or a bi-fimctional (e.g. bi-specific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In one aspect, the antibodies and fragments thereof of the invention binds a tumor-associated antigen protein with wild-type or enhanced affinity.

[0175] Also provided herein are methods for obtaining an antibody antigen binding domain specific for a target antigen, the method comprising providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a V _H (or V _H H) domain set out herein a V _H domain which is an amino acid sequence variant of the V _H domain, optionally combining the V _H domain thus provided with one or more V _L domains, and testing the V _H domain or V _H /V _L combination or combinations to identify a specific binding member or an antibody antigen binding domain specific for a target antigen of interest and optionally with one or more desired properties.

[0176] In some instances, VH domains, VHH domains, and scFvs can be prepared according to method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). scFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The linker length can greatly affect how the variable regions of a scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5-10 amino acids) intra-chain folding is prevented. Inter-chain folding is also required to bring the two variable regions together to form a functional epitope binding site. In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G4S)n, wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S)n, wherein n=l to 3. For examples of linker orientation and size see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S.

Patent Application Publication Nos. 20050100543 and 20050175606, U.S. Patent No. 7,695,936, and PCT publication Nos. W02006020258 and W02007024715, each of which is incorporated herein by reference.

Linkers for Antibody and/or Blocking Domains

[0177] Blocking of biological activity must be reversible for the molecule to be useful. Blocking can be removed by removal of the sterically blocking domain. In one embodiment, the blocking domain is removed by proteolytic cleavage of the linker by which the blocking domain is attached to the TFP T cell. In another embodiment, the blocking domain is removed by proteolytic cleavage of the linker by which an anti-blocking domain antibody (e.g., an anti-HSA antibody) is removed by proteolytic cleavage of the linker by which the blocking domain is attached to the TFP T cell. In an aspect, the blocking domain is removed by enzymatic cleavage other than proteolytic cleavage. A feature of this invention is that removal of the blocking domain is enzymatic and not merely a function of elapsed time. In an aspect, the blocking domain is removed at the site of disease by an enzyme overproduced in diseased tissue. In some embodiments, the domain is removed in a tumor environment by a protease produced by tumor cells. In some embodiments, the protease -cleavable linker comprises a sequence recognized by a protease produced by tumor cells in the tumor microenvironment (TME). In one embodiment, a "blocked" TFP T cell, i.e., a TFP T cell having a blocking domain bound by a protease-cleavable linker or a TFP T cell having a blocking domain bound by an binding moiety (e.g., an antibody), will remain blocked until reaching the TME, i.e., will be in the presence of a protease that will recognize and cleave the linker that keeps the blocking domain tethered to the TFP T cell.

[0178] In some embodiments, the protease is at least one of a tumor cell surface protease, a

carboxypeptidase, a cathepsin, a kallikrein, a hexokinase, a plasmin, a stromelysin, factor Xa, a chymotrypsin-like protease, a trypsin-like protease, a elastase-like protease, a tryptase, a chymase, a subtilisin-like protease, an actinidain, a proteinase, a bromelain, a calpain, a caspase, a cysteine protease, a papain, an FHV-l protease, an HSV protease, a CMV protease, a chymosin, a renin, a pepsin, a matriptase, a plasmepsin, a nepenthesin, a metalloexopeptidase, a metalloendopeptidase, a matrix metalloproteinase/ collagenase, a plasminogen activator, a urokinase plasminogen activator (uPA), an enterokinase, a prostate-specific antigen (PSA, hK3), an interleukin- 1 b converting enzyme, a thrombin, a fibroblast activation protein (FAP), a meprin, a granzyme, and a dipeptidyl peptidase. In some embodiments, the cathepsin is cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, or cathepsin F; the hexokinase is hKl, hKlO, or hKl5; the proteinase is PR-3; the caspase is caspase-3; the cysteine protease is Mir 1 -CP or legumain; the matrix metalloproteinase or collagenase is

MMP l/interstitial collagenase, MMP2/type IV collagenase, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP 12, MMP 13, MMP 14, MMP15, MMP 16, ADAM 10, or ADAM 12; the prostate -specific antigen is PSA or hK3, the FAP is FAP-a: the granzyme is granzyme M or granzyme B; or the dipeptidyl peptidase is dipeptidyl peptidase IV (DPPIV/CD26). In some embodiments, more than one proteolytic cleavage site is present.

[0179] In some embodiments, the linker of the blocking domain comprises a protease-cleavable linker. The protease-cleavable linker is engineered to comprise a sequence recognized by a protease typically present in the tumor microenvironment (TME). In some embodiments, the protease-cleavable linker is cleavable by a matrix metalloprotease (MMP). Examples of MMPs include MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP 10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP 16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27. In some embodiments, the protease- cleavable linker is a substrate for MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. In some embodiments, the protease-cleavable linker is a substrate for MMP9. In some embodiments, the protease-cleavable linker is a substrate for MMP 14. In some embodiments, the protease-cleavable linker is a substrate for two or more MMPs. In some embodiments, the protease- cleavable linker is a substrate for at least MMP9 and MMP14. In some embodiments, the protease- cleavable linker comprises two or more substrates for the same MMP. In some embodiments, the protease-cleavable linker comprises at least two or more MMP9 substrates. In some embodiments, the protease-cleavable linker comprises at least two or more MMP14 substrates. In some embodiments, the protease-cleavable linker is a substrate for an MMP and includes the sequence ISSGFFSS (SEQ ID NO:43); QNQAFRMA (SEQ ID NO:44); AQNLLGMV (SEQ ID NO:45); STFPFGMF (SEQ ID NO:46); PVGYTSSL (SEQ ID NO:47); DWLYWPGI (SEQ ID NO:48); MIAPVAYR (SEQ ID NO:49); RPSPMWAY (SEQ ID NO:50); WATPRPMR (SEQ ID NO:5 l); FRLLDWQW (SEQ ID NO:52); LKAAPRWA (SEQ ID NO:53); GPSHLVLT (SEQ ID NO:54); LPGGLSPW (SEQ ID NO:55);

MGLFSEAG (SEQ ID NO:56); SPLPLRVP (SEQ ID NO:57); RMHLRSLG (SEQ ID NO:58);

LAAPLGLL (SEQ ID NO:59); AVGLLAPP (SEQ ID NO:60); LLAPSHRA (SEQ ID NO:6l);

PAGLWLDP (SEQ ID NO:62); and/or ISSGLSS (SEQ ID NO:63).

Table 1. MMP Cleavable Core Consensus Sequence 1

R, or W; X ₂₈ is A, D, or G; and X ₂₉ is C or Y.

(SEQ ID NO: 70)

Table 2. MMP9 Cleavable Core Consensus Sequence 2

Table 3. MMP9 Cleavable Core Consensus Sequence 3

Table 4. MMP9 Cleavable Core Consensus Sequence _ _

Table 5. MMP14 Cleavable Core Consensus Sequence 5

_ _

Table 6. MMP14 Cleavable Core Consensus Sequence 6

Table 6A. MMP14 Cleavable Core Consensus Sequence 6A

Table 7. MMP14 Cleavable Core Consensus Sequence 7

Table 8. MMP14 Cleavable Core Consensus Sequence 8

Table 8A. MMP14 Cleavable Core Consensus Sequence 8 A

Table 9. MMP14 Cleavable Core Consensus Sequence 9 _ _

Table 10. MMP14 Cleavable Core Consensus Sequence 10

_ _

Table 11. MMP14 Cleavable Core Consensus Sequence 11

Table 12. MMP14 Cleavable Core Consensus Sequence 12

_

Table 13. MMP14 Cleavable Core Consensus Sequence 13

Core Consensus 13 Subgenus of Core Consensus 13

Stability and Mutations

[0180] The stability of an anti-tumor-associated antigen binding domain, e.g., scFv molecules (e.g., soluble scFv) can be evaluated in reference to the biophysical properties (e.g., thermal stability) of a conventional control scFv molecule or a full-length antibody. In one embodiment, the humanized or human scFv has a thermal stability that is greater than about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees Celsius than a parent scFv in the described assays.

[0181] The improved thermal stability of the anti-tumor-associated antigen binding domain, e.g., scFv is subsequently conferred to the entire tumor-associated antigen-TFP construct, leading to improved therapeutic properties of the anti-tumor-associated antigen TFP construct. The thermal stability of the anti-tumor-associated antigen binding domain, e.g., an scFv or sdAb, can be improved by at least about 2 ° C or 3 ° C as compared to a conventional antibody. In one embodiment, the anti -tumor-associated antigen binding domain, e.g., an scFv or sdAb, has a 1 ° C improved thermal stability as compared to a conventional antibody. In another embodiment, the anti-tumor-associated antigen binding domain, e.g., an scFv or sdAb, has a 2 ° C improved thermal stability as compared to a conventional antibody. In another embodiment, the scFv has a 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C, 10 ° C, 11 ° C, 12 ° C, 13 ° C, 14 ° C, or 15 ° C improved thermal stability as compared to a conventional antibody. Comparisons can be made, for example, between the scFv molecules disclosed herein and scFv molecules or Fab fragments of an antibody from which the scFv VH and VL were derived. Thermal stability can be measured using methods known in the art. For example, in one embodiment, TM can be measured. Methods for measuring TM and other methods of determining protein stability are described below.

[0182] Mutations in an scFv or an sdAb (arising through humanization or mutagenesis of the soluble scFv or sdAb) alter the stability of the scFv and improve the overall stability of the scFv and the anti- tumor-associated antigen TFP construct. Stability of the humanized scFv is compared against the murine scFv using measurements such as TM, temperature denaturation and temperature aggregation. In one embodiment, the anti-tumor-associated antigen binding domain, e.g., a scFv, comprises at least one mutation arising from the humanization process such that the mutated scFv confers improved stability to the anti-tumor-associated antigen TFP construct. In another embodiment, the anti-tumor-associated antigen binding domain, e.g., scFv comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations arising from the humanization process such that the mutated scFv confers improved stability to the tumor-associated antigen-TFP construct.

[0183] In one aspect, the antigen binding domain of the TFP comprises an amino acid sequence that is homologous to an antigen binding domain amino acid sequence described herein, and the antigen binding domain retains the desired functional properties of the anti-tumor-associated antigen antibody fragments described herein. In one specific aspect, the TFP composition of the invention comprises an antibody fragment. In a further aspect, that antibody fragment comprises a scFv.

[0184] In various aspects, the antigen binding domain of the TFP is engineered by modifying one or more amino acids within one or both variable regions (e.g., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions. In one specific aspect, the TFP composition of the invention comprises an antibody fragment. In a further aspect, that antibody fragment comprises a scFv.

[0185] It will be understood by one of ordinary skill in the art that the antibody or antibody fragment of the invention may further be modified such that they vary in amino acid sequence (e.g., from wild-type), but not in desired activity. For example, additional nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues may be made to the protein. For example, a nonessential amino acid residue in a molecule may be replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members, e.g., a conservative substitution, in which an amino acid residue is replaced with an amino acid residue having a similar side chain, may be made.

[0186] Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0187] Percent identity in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 70%, 71% , 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0188] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat' 1. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Brent et ah, (2003) Current Protocols in Molecular Biology). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et ah, (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et ah, (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0189] In one aspect, the present invention contemplates modifications of the starting antibody or fragment (e.g., scFv) amino acid sequence that generate functionally equivalent molecules. For example, the VH or VL of an anti-tumor-associated antigen binding domain, e.g., scFv, comprised in the TFP can be modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting VH or VL framework region of the anti -tumor-associated antigen binding domain, e.g., scFv. The present invention contemplates modifications of the entire TFP construct, e.g., modifications in one or more amino acid sequences of the various domains of the TFP construct in order to generate functionally equivalent molecules. The TFP construct can be modified to retain at least about

70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,

88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity of the starting TFP construct.

[0190] Linkers for attachment of TAA binding domains and a TCR protein

[0191] Optionally, a short oligo- or polypeptide linker, between 2 and 20 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic region of the TFP. A glycine - serine doublet provides a particularly suitable linker. For example, in one aspect, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 181). In some embodiments, the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 182). In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGGSGGGGSLE (SEQ ID NO: 183). In other embodiments, the linker comprises the amino acid sequence of

AAAGGGGSGGGGSGGGGSLE (SEQ ID NO: 184). In other embodiments, the linker is a long linker having the sequence AAIEVMYPPPYLGGGGSGGGGSGGGGSLE (SEQ ID NO: 185). In some embodiments, the linker is encoded by a nucleotide sequence of

GGTGGAGGCGGTTCTGGTGGAGGCGGTTCGGATGGCGGAGGTTCA (SEQ ID NO: 186). In other embodiments, the linker is encoded by a nucleotide sequence of

GGAGAGGGTAAATCTTCCGGATCTGGTTCCGAAAGCAAGGCTAGC (SEQ ID NO: 187).

Extracellular domain

[0192] The extracellular domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any protein, but in particular a membrane- bound or transmembrane protein. In one aspect, the extracellular domain is capable of associating with the transmembrane domain. An extracellular domain of particular use in this invention may include at least the extracellular region(s) of e.g., the alpha, beta or zeta chain of the T cell receptor, or CD3 epsilon, CD3 gamma, or CD3 delta, or in alternative embodiments, CD28, CD45, CD2, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

Transmembrane Domain

[0193] In general, a TFP sequence contains an extracellular domain and a transmembrane domain encoded by a single genomic sequence. In alternative embodiments, a TFP can be designed to comprise a transmembrane domain that is heterologous to the extracellular domain of the TFP. A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids of the intracellular region). In some cases, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the extracellular region. In some cases, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the intracellular region. In one aspect, the transmembrane domain is one that is associated with one of the other domains of the TFP is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerization with another TFP on the TFP T cell surface. In a different aspect the amino acid sequence of the

transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same TFP.

[0194] The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane -bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TFP has bound to a target. A transmembrane domain of particular use in this invention may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0195] In some instances, the transmembrane domain can be attached to the extracellular region of the TFP, e.g. , the antigen binding domain of the TFP, via a hinge, e.g. , a hinge from a human protein. For example, in one embodiment, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge.

Cytoplasmic Domain

[0196] The cytoplasmic domain of the TFP can include an intracellular signaling domain, if the TFP contains CD3 gamma, delta or epsilon polypeptides; TCR alpha and TCR beta subunits are generally lacking in a signaling domain. An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the TFP has been introduced. The term "effector function" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0197] Examples of intracellular signaling domains for use in the TFP of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0198] It is known that signals generated through the TCR alone are insufficient for full activation of naive T cells and that a secondary and/or costimulatory signal is required. Thus, naive T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen -independent manner to provide a secondary or costimulatory signal

(secondary cytoplasmic domain, e.g., a costimulatory domain).

[0199] A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine -based activation motifs (ITAMs).

[0200] Examples of ITAMs containing primary intracellular signaling domains that are of particular use in the invention include those of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, a TFP of the invention comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-epsilon. In one embodiment, a primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain. In one embodiment, a primary signaling domain comprises a modified ITAM -containing primary intracellular signaling domain, e.g., an optimized and/or truncated ITAM -containing primary intracellular signaling domain. In an embodiment, a primary signaling domain comprises one, two, three, four or more ITAM motifs.

[0201] The intracellular signaling domain of the TFP can comprise the CD3 zeta signaling domain by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a TFP of the invention. For example, the intracellular signaling domain of the TFP can comprise a CD3 epsilon chain portion and a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the TFP comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human TFP T cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3):696-706).

[0202] The intracellular signaling sequences within the cytoplasmic portion of the TFP of the invention may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences.

[0203] In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable linker.

[0204] In one aspect, the TFP-expressing cell described herein can further comprise a second TFP, e.g., a second TFP that includes a different antigen binding domain, e.g., to the same target (e.g., CD22) or a different target (e.g., CD123). In one embodiment, when the TFP-expressing cell comprises two or more different TFPs, the antigen binding domains of the different TFPs can be such that the antigen binding domains do not interact with one another. For example, a cell expressing a first and second TFP can have an antigen binding domain of the first TFP, e.g., as a fragment, e.g., a scFv, that does not associate with the antigen binding domain of the second TFP, e.g., the antigen binding domain of the second TFP is a VHH.

[0205] In another aspect, the TFP-expressing cell described herein can further express another agent, e.g., an agent which enhances the activity of a TFP-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., PD 1, can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 and TGFR beta. In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4 and TIGIT, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of an extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein). PD1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA. PD-l is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD- L2 have been shown to downregulate T cell activation upon binding to PD1 (Freeman et al. 2000 J Exp Med 192: 1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634- 43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.

[0206] In one embodiment, the agent comprises the extracellular domain (ECD) of an inhibitory molecule, e.g., Programmed Death 1 (PD1) can be fused to a transmembrane domain and optionally an intracellular signaling domain such as 41BB and CD3 zeta (also referred to herein as a PD1 TFP). In one embodiment, the PD1 TFP, when used in combinations with an anti -tumor antigen TFP described herein, improves the persistence of the T cell. In one embodiment, the TFP is a PD1 TFP comprising the extracellular domain of PD 1. Alternatively, provided are TFPs containing an antibody or antibody fragment such as a scFv that specifically binds to the Programmed Death -Ligand 1 (PD-L1) or

Programmed Death-Ligand 2 (PD-L2).

[0207] In another aspect, the present invention provides a population of TFP -expressing T cells, e.g., TFP T cells. In some embodiments, the population of TFP-expressing T cells comprises a mixture of cells expressing different TFPs. For example, in one embodiment, the population of TFP T cells can include a first cell expressing a TFP having an anti-tumor-associated antigen binding domain described herein, and a second cell expressing a TFP having a different anti-tumor-associated antigen binding domain, e.g., an anti-tumor-associated antigen binding domain described herein that differs from the anti- tumor-associated antigen binding domain in the TFP expressed by the first cell. As another example, the population of TFP-expressing cells can include a first cell expressing a TFP that includes an anti -tumor- associated antigen binding domain, e.g., as described herein, and a second cell expressing a TFP that includes an antigen binding domain to a target other than tumor-associated antigen (e.g., another tumor- associated antigen).

[0208] In another aspect, the present invention provides a population of cells wherein at least one cell in the population expresses a TFP having an anti-tumor-associated antigen domain described herein, and a second cell expressing another agent, e.g., an agent which enhances the activity of a TFP-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule.

Inhibitory molecules, e.g., can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. In one

embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. [0209] Disclosed herein are methods for producing in vitro transcribed RNA encoding TFPs. The present invention also includes a TFP encoding RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3 ' and 5 ' untranslated sequence ("UTR"), a 5 ' cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length. RNA so produced can efficiently transfect different kinds of cells. In one aspect, the template includes sequences for the TFP.

[0210] In one aspect, the anti-TAA TFP is encoded by a circular RNA comprising a sequence encoding a TFP, a CAR, a TCR or combination thereof. In some embodiments, circular RNA is exogenous. In other embodiments, circular RNA is endogenous. In other embodiments, circular RNAs with an internal ribosomal entry site (IRES) can be translated in vitro or ex vivo.

[0211] Circular RNAs (circular RNAs) are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Circular RNAs are 3-5 ' covalently closed RNA rings, and circular RNAs do not display Cap or poly(A) tails. Since circular RNAs lack the free ends necessary for exonuclease-mediated degradation, rendering them resistant to several mechanisms of RNA turnover and granting them extended lifespans as compared to their linear mRNA counterparts. For this reason, circularization may allow for the stabilization of mRNAs that generally suffer from short serum half-lives and may therefore improve the overall efficacy of mRNA in a variety of applications. Circular RNAs are produced by the process of splicing, and circularization occurs using conventional splice sites mostly at annotated exon boundaries (Starke et ah, 2015; Szabo et ah, 2015). For circularization, splice sites are used in reverse: downstream splice donors are "backspliced" to upstream splice acceptors (see Jeck and Sharpless, 2014; Barrett and Salzman, 2016; Szabo and Salzman, 2016; Holdt et ah, 2018 for review).

[0212] To generate circular RNAs for subsequently transfer into cells, in vitro production of circular RNAs with autocatalytic-splicing introns can be programmed. In one embodiment, the method for generating circular RNA comprises in vitro transcription (IVT) of a precursor linear RNA template with specially designed primers. Three general strategies have been reported so far for RNA circularization: chemical methods using cyanogen bromide or a similar condensing agent; enzymatic methods using RNA or DNA ligases; and ribozymatic methods using self-splicing introns. In some embodiments, precursor RNA is synthesized by run-off transcription and then heated in the presence of magnesium ions and GTP to promote circularization. RNA so produced can efficiently transfect different kinds of cells.

In some exemplary embodiments, PCR is used to generate a template for in vitro transcription of linear precursor RNA which is used for transfection. Methods for performing PCR are well known in the art.

[0213] In one aspect the anti-TAA TFP is encoded by a circular RNA. In one embodiment, the circular RNA encoding the anti-TAA TFP is introduced into a T cell for production of a TFP T cell. In one embodiment, the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection. In one embodiment, the circular RNA encoding the anti-TAA TFP is introduced to a subject via a targeted nanoparticle. [0214] In one aspect, the anti-TAA TFP is encoded by a messenger RNA (mRNA). In one aspect, the mRNA encoding the anti-tumor-associated antigen TFP is introduced into a T cell for production of a TFP T cell. In one embodiment, the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection. The RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. The desired template for in vitro transcription is a TFP of the present invention. In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the nucleic acid can include some or all of the 5 ' and/or 3 ' untranslated regions (UTRs). The nucleic acid can include exons and introns. In one embodiment, the DNA to be used for PCR is a human nucleic acid sequence. In another embodiment, the DNA to be used for PCR is a human nucleic acid sequence including the 5 ' and 3 ' UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.

[0215] PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR. "Substantially complementary," as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR. The primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5 ' and 3 ' UTRs. The primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5 ' and 3 ' UTRs. Primers useful for PCR can be generated by synthetic methods that are well known in the art. "Forward primers" are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified. "Upstream" is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand. "Reverse primers" are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified.

"Downstream" is used herein to refer to a location 3 ' to the DNA sequence to be amplified relative to the coding strand. [0216] Any DNA polymerase useful for PCR can be used in the methods disclosed herein. The reagents and polymerase are commercially available from a number of sources.

[0217] Chemical structures with the ability to promote stability and/or translation efficiency may also be used. The RNA preferably has 5 ' and 3 ' UTRs. In one embodiment, the 5 ' UTR is between one and 3,000 nucleotides in length. The length of 5 ' and 3 ' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5 ' and 3 ' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

[0218] The 5 ' and 3 ' UTRs can be the naturally occurring, endogenous 5 ' and 3 ' UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3 ' UTR sequences can decrease the stability of mRNA. Therefore, 3 ' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[0219] In one embodiment, the 5 ' UTR can contain the Kozak sequence of the endogenous nucleic acid. Alternatively, when a 5 ' UTR that is not endogenous to the nucleic acid of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5 ' UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5 ' UTR can be 5 ' UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogues can be used in the 3 ' or 5 ' UTR to impede exonuclease degradation of the mRNA.

[0220] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5 ' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0221] In a preferred embodiment, the mRNA has both a cap on the 5 ' end and a 3 ' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3 ' UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.

[0222] On a linear DNA template, phage T7 RNA polymerase can extend the 3 ' end of the transcript beyond the last base of the template (Schenbom and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003).

[0223] The conventional method of integration of polyA/T stretches into a DNA template is molecular cloning. However, polyA/T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable. That is why a method which allows construction of DNA templates with polyA/T 3 ' stretch without cloning highly desirable.

[0224] The polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100 T tail (size can be 50-5000 Ts), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.

[0225] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly (A) polymerase, such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3 ' end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.

[0226] 5 ' caps on also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods disclosed herein include a 5 ' cap. The 5 ' cap is provided using techniques known in the art and described herein (Cougot, et ah, Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et ah, RNA, 7: 1468-95 (2001); Elango, et ak, Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0227] The RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.

[0228] RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa

Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as "gene guns" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8): 861 -70 (2001).

Nucleic Acid Constructs Encoding a TFP

[0229] The present invention also provides nucleic acid molecules encoding one or more TFP constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct.

[0230] The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[0231] The present invention also provides vectors in which a DNA of the present invention is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco -retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0232] In another embodiment, the vector comprising the nucleic acid encoding the desired TFP of the invention is an adenoviral vector (A5/35).

Modified T cells

[0233] Disclosed herein, in some embodiments, are modified T cells comprising the recombinant nucleic acid disclosed herein, or the vectors disclosed herein, wherein the modified T cell comprises a functional disruption of an endogenous TCR. Also disclosed herein, in some embodiments, are modified T cells comprising the sequence encoding the TFP of the nucleic acid disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein, wherein the modified T cell comprises a functional disruption of an endogenous TCR. Further disclosed herein, in some embodiments, are allogenic modified T cells comprising the sequence encoding the inducible TFP disclosed herein or an inducible TFP encoded by the sequence of the nucleic acid disclosed herein.

In some instances, the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain. In some instances, the endogenous TCR that is functionally disrupted is an endogenous TCR alpha chain, an endogenous TCR beta chain, or an endogenous TCR alpha chain and an endogenous TCR beta chain. In some instances, the endogenous TCR that is functionally disrupted has reduced binding to MHC -peptide complex compared to that of an unmodified control T cell. In some instances, the functional disruption is a disruption of a gene encoding the endogenous TCR. In some instances, the disruption of a gene encoding the endogenous TCR is a removal of a sequence of the gene encoding the endogenous TCR from the genome of a T cell. In some instances, the T cell is a human T cell. In some instances, the T cell is a CD8+ or CD4+ T cell or CD4+CD8+ T cell. In some instances, the T cell is an allogenic T cell. In some instances, the modified T cells further comprise a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain. In some instances, the inhibitory molecule comprises the first polypeptide comprising at least a portion of PD 1 and the second polypeptide comprising a costimulatory domain and primary signaling domain. Methods of Producing Modified T cells

[0234] Disclosed herein, in some embodiments, are methods of producing the modified T cell of the disclosure, the method comprising (a) disrupting an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, or any combination thereof; thereby producing a T cell containing a functional disruption of an endogenous TCR gene; and (b) transducing the T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid of the disclosure, or the vectors disclosed herein. In some instances, disrupting comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets the endogenous gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.

[0235] Further disclosed herein, in some embodiments, are methods of producing the modified T cell of the disclosure, the method comprising transducing a T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid disclosed herein, or the vectors disclosed herein. In some instances, the T cell containing a functional disruption of an endogenous TCR gene is a T cell containing a functional disruption of an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.

[0236] In some instances, the T cell is a human T cell. In some instances, the T cell containing a functional disruption of an endogenous TCR gene has reduced binding to MHC-peptide complex compared to that of an unmodified control T cell.

[0237] In some instances, the nuclease is a meganuclease, a zinc -finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, CRISPR/Cas nickase, or a megaTAL nuclease. In some instances, the sequence comprised by the recombinant nucleic acid or the vector is inserted into the endogenous TCR subunit gene at the cleavage site, and wherein the insertion of the sequence into the endogenous TCR subunit gene functionally disrupts the endogenous TCR subunit. In some instances, the nuclease is a meganuclease. In some instances, the meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence, and wherein the second subunit binds to a second recognition half-site of the recognition sequence. In some instances, the meganuclease is a single chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.

Gene Editing Technologies

[0238] In some embodiments, the modified T cells disclosed herein are engineered using a gene editing technique such as clustered regularly interspaced short palindromic repeats (CRISPR®, see, e.g., US

Patent No. 8,697,359), transcription activator-like effector (TALE) nucleases (TALENs, see, e.g., U.S. Patent No. 9,393,257), meganucleases (endodeoxyribonucleases having large recognition sites comprising double -stranded DNA sequences of 12 to 40 base pairs), zinc finger nuclease (ZFN, see, e.g., Umov et al., Nat. Rev. Genetics (2010) vl 1, 636-646), or megaTAL nucleases (a fusion protein of a meganuclease to TAL repeats) methods. In this way, a chimeric construct may be engineered to combine desirable characteristics of each subunit, such as conformation or signaling capabilities. See also Sander & Joung, Nat. Biotech. (2014) v32, 347-55; and June et al., 2009 Nature Reviews Immunol. 9.10: 704- 716, each incorporated herein by reference. In some embodiments, one or more of the extracellular domain, the transmembrane domain, or the cytoplasmic domain of a TFP subunit are engineered to have aspects of more than one natural TCR subunit domain (i.e., are chimeric).

[0239] Recent developments of technologies to permanently alter the human genome and to introduce site-specific genome modifications in disease relevant genes lay the foundation for therapeutic applications. These technologies are now commonly known as "genome editing".

[0240] In some embodiments, gene editing techniques are employed to disrupt an endogenous TCR gene. In some embodiments, mentioned endogenous TCR gene encodes a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain. In some embodiments, gene editing techniques pave the way for multiplex genomic editing, which allows simultaneous disruption of multiple genomic loci in endogenous TCR gene. In some embodiments, multiplex genomic editing tecniques are applied to generate gene-disrupted T cells that are deficient in the expression of endogenous TCR, and/or human leukocyte antigens (HLAs), and/or programmed cell death protein 1 (PD1), and/or other genes.

[0241] Current gene editing technologies comprise meganucleases, zinc -finger nucleases (ZFN), TAL effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats

(CRISPR)/CRISPR-associated (Cas) system. These four major classes of gene-editing techniques share a common mode of action in binding a user-defined sequence of DNA and mediating a double-stranded DNA break (DSB). DSB may then be repaired by either non -homologous end joining (NHEJ) or -when donor DNA is present- homologous recombination (HR), an event that introduces the homologous sequence from a donor DNA fragment. Additionally, nickase nucleases generate single -stranded DNA breaks (SSB). DSBs may be repaired by single strand DNA incorporation (ssDI) or single strand template repair (ssTR), an event that introduces the homologous sequence from a donor DNA.

[0242] Genetic modification of genomic DNA can be performed using site -specific, rare-cutting endonucleases that are engineered to recognize DNA sequences in the locus of interest. Methods for producing engineered, site-specific endonucleases are known in the art. For example, zinc -finger nucleases (ZFNs) can be engineered to recognize and cut predetermined sites in a genome. ZFNs are chimeric proteins comprising a zinc finger DNA-binding domain fused to the nuclease domain of the Fokl restriction enzyme. The zinc finger domain can be redesigned through rational or experimental means to produce a protein that binds to a pre-determined DNA sequence -18 basepairs in length. By fusing this engineered protein domain to the Fokl nuclease, it is possible to target DNA breaks with genome-level specificity. ZFNs have been used extensively to target gene addition, removal, and substitution in a wide range of eukaryotic organisms (reviewed in Durai et al. (2005), Nucleic Acids Res 33, 5978). Likewise, TAL-effector nucleases (TALENs) can be generated to cleave specific sites in genomic DNA. Like a ZFN, a TALEN comprises an engineered, site-specific DNA-binding domain fused to the Fokl nuclease domain (reviewed in Mak et al. (2013), Curr Opin Struct Biol. 23:93-9). In this case, however, the DNA binding domain comprises a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA base pair. Compact TALENs have an alternative

endonuclease architecture that avoids the need for dimerization (Beurdeley et al. (2013), Nat Commun. 4: 1762). A Compact TALEN comprises an engineered, site -specific TAL-effector DNA-binding domain fused to the nuclease domain from the I-Tevl homing endonuclease. Unlike Fokl, I-Tevl does not need to dimerize to produce a double-strand DNA break so a Compact TALEN is functional as a monomer.

[0243] Engineered endonucleases based on the CRISPR/Cas9 system are also known in the art (Ran et al. (2013), Nat Protoc. 8:2281-2308; Mali et al. (2013), Nat Methods 10:957-63). The CRISPR gene editing technology is composed of an endonuclease protein whose DNA-targeting specificity and cutting activity can be programmed by a short guide RNA or a duplex crRNA/TracrRNA. A CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically microbial Cas9; and (2) a short "guide RNA" or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome. By expressing multiple guide RNAs in the same cell, each having a different targeting sequence, it is possible to target DNA breaks simultaneously to multiple sites in the genome (multiplex genomic editing).

[0244] There are two classes of CRISPR systems known in the art (Adli (2018) Nat. Commun. 9: 191 1), each containing multiple CRISPR types. Class 1 contains type I and type III CRISPR systems that are commonly found in Archaea. And, Class II contains type II, IV, V, and VI CRISPR systems. Although the the most widely used CRISPR/Cas system is the the type II CRISPR-Cas9 system, CRISPR/Cas systems have been repurposed by researchers for genome editing. More than 10 different CRISPR/Cas proteins have been remodeled within last few years (Adli (2018) Nat. Commun. 9: 1911). Among these, such as Casl2a (Cpfl) proteins from Acid- aminococcus sp (AsCpfl) and Lachnospiraceae bacterium (LbCpfl), are particularly interesting.

[0245] Homing endonucleases are a group of naturally occurring nucleases that recognize 15-40 base- pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as group 1 self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by producing a double -stranded break in the chromosome, which recruits the cellular DNA-repair machinery (Stoddard (2006), Q. Rev. Biophys. 38: 49-95). Specific amino acid substations could reprogram DNA cleavage specificity of homing nucleases (Niyonzima (2017), Protein Eng Des Sel. 30(7): 503-522).

Meganucleases (MN) are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases and engineered for a unique target site (Gersbach (2016), Molecular Therapy. 24: 430-446). In some embodiments, meganuclease is engineered I-Crel homing endonuclease. In other embodiments, meganuclease is engineered I-Scel homing endonuclease.

[0246] In addition to mentioned four major gene editing technologies, chimeric proteins comprising fusions of meganucleases, ZFNs, and TALENs have been engineered to generate novel monomeric enzymes that take advantage of the binding affinity of ZFNs and TALENs and the cleavage specificity of meganucleases (Gersbach (2016), Molecular Therapy. 24: 430-446). For example, A megaTAL is a single chimeric protein, which is the combination of the easy-to-tailor DNA binding domains from TALENs with the high cleavage efficiency of meganucleases.

[0247] In order to perform the gene editing technique, the nucleases, and in the case of the CRISPR/ Cas9 system, a gRNA, must be efficiently delivered to the cells of interest. Delivery methods such as physical, chemical, and viral methods are also know in the art (Mali (2013). Indian J. Hum. Genet. 19: 3- 8.). In some instances, physical delivery methods can be selected from the methods but not limited to electroporation, microinjection, or use of ballistic particles. On the other hand, chemical delivery methods require use of complex molecules such calcium phosphate, lipid, or protein. In some embodiments, viral delivery methods are applied for gene editing techniques using viruses such as but not limited to adenovirus, lentivirus, and retrovirus.

[0248] The expression constructs of the present invention may also be used for nucleic acid

immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art (see, e.g., U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties). In another embodiment, the invention provides a gene therapy vector.

[0249] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0250] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, e.g., in Sambrook et al., 2012, Molecular Cloning:

A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

[0251] A number of virally based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[0252] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0253] An example of a promoter that is capable of expressing a TFP transgene in a mammalian T cell is the EFla promoter. The native EFla promoter drives expression of the alpha subunit of the elongation factor-l complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EFla promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving TFP expression from transgenes cloned into a lentiviral vector (see, e.g., Milone et ah, Mol. Ther. 17(8): 1453-1464 (2009)). Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human

immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor- la promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline -regulated promoter.

[0254] In order to assess the expression of a TFP polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic -resistance genes, such as neo and the like.

[0255] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5 ' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.

[0256] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0257] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art (see, e.g., Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection

[0258] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like (see, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362).

[0259] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.

[0260] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0261] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 ° C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. "Liposome" is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates.

Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al, 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine -nucleic acid complexes.

[0262] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0263] The present invention further provides a vector comprising a TFP encoding nucleic acid molecule. In one aspect, a TFP vector can be directly transduced into a cell, e.g., a T cell. In one aspect, the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs. In one aspect, the vector is capable of expressing the TFP construct in mammalian T cells. In one aspect, the mammalian T cell is a human T cell. Sources of T cells

[0264] Prior to expansion and genetic modification, a source of T cells is obtained from a subject. The term "subject" is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects of the present invention, any number of T cell lines available in the art, may be used. In certain aspects of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll® separation. In one preferred aspect, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.

In one aspect, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one aspect of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative aspect, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi -automated "flow-through" centrifuge (for example, the COBE® 2991 cell processor, the Baxter CytoMate®, or the Haemonetics® Cell Saver® 5) according to the manufacturer' s instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte® A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture medium.

[0265] In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a Percoll® gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one aspect, T cells are isolated by incubation with anti-CD3/anti-CD28 (e.g., 3x28)-conjugated beads, such as Dynabeads® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In one aspect, the time period is about 30 minutes. In a further aspect, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from

immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention. In certain aspects, it may be desirable to perform the selection procedure and use the "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to further rounds of selection.

[0266] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8. In certain aspects, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain aspects, T regulatory cells are depleted by anti- C25 conjugated beads or other similar method of selection.

[0267] In one embodiment, a T cell population can be selected that expresses one or more of IFN-g, TNF-alpha, IF-17A, IF-2, IF-3, IF-4, GM-CSF, IF-10, IF-13, granzyme B, and perforin, or other appropriate molecules, e.g., other cytokines. Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No.: WO2013/126712.

[0268] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain aspects, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (e.g., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one aspect, a concentration of 2 billion cells/mF is used. In one aspect, a concentration of 1 billion cells/mF is used. In a further aspect, greater than 100 million cells/mF is used. In a further aspect, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mF is used. In yet one aspect, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mF is used. In further aspects, concentrations of 125 or 150 million cells/mF can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[0269] In a related aspect, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one aspect, the concentration of cells used is 5 x l0 ⁶ /mL. In other aspects, the concentration used can be from about 1 x l0 ⁵ /mL to 1 x l0 ⁶ /mL, and any integer value in between. In other aspects, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10 ° C or at room temperature.

[0270] T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25%

Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan® and PlasmaLyte® A, the cells then are frozen to -80 ° C at a rate of 1 per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20 ° C or in liquid nitrogen. In certain aspects, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present invention.

[0271] Also contemplated in the context of the invention is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one aspect, a blood sample or an apheresis is taken from a generally healthy subject. In certain aspects, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain aspects, the T cells may be expanded, frozen, and used at a later time. In certain aspects, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further aspect, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and tacrolimus (FK506), antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporin, rapamycin, mycophenolic acid, steroids, romidepsin (formerly FR901228), and irradiation.

[0272] In a further aspect of the present invention, T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present invention to collect blood cells, including T cells, dendritic cells, or other cells of the

hematopoietic lineage, during this recovery phase. Further, in certain aspects, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

Activation and Expansion of T Cells

[0273] T cells may be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631.

[0274] Generally, the T cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co -stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti- CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et ah, Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol. Meth. 227(l-2):53-63, 1999).

[0275] T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.

[0276] Further, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.

[0277] Once an anti-tumor-associated antigen TFP is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following antigen stimulation, sustain T cell expansion in the absence of re -stimulation, and anti-cancer activities in appropriate in vitro and animal models. Assays to evaluate the effects of an anti-tumor-associated antigen TFP are described in further detail below

[0278] Western blot analysis of TFP expression in primary T cells can be used to detect the presence of monomers and dimers (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). In some embodiments, T cells (1: 1 mixture of CD4+ and CD8+ T cells) expressing the TFPs are expanded in vitro for more than 10 days followed by lysis and SDS-PAGE under reducing conditions. TFPs are detected by western blotting using an antibody to a TCR chain. The same T cell subsets are used for SDS-PAGE analysis under non-reducing conditions to permit evaluation of covalent dimer formation.

[0279] In vitro expansion of TFP+ T cells following antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4+ and CD8+ T cells are stimulated with alphaCD3/alphaCD28 and APCs followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed. Exemplary promoters include the CMV IE gene, EF-lalpha, ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescence is evaluated on day 6 of culture in the CD4+ and/or CD8+ T cell subsets by flow cytometry (see, e.g., Milone et al., Molecular Therapy 17(8): 1453 - 1464 (2009)). Alternatively, a mixture of CD4+ and CD8+ T cells are stimulated with

alphaCD3/alphaCD28 coated magnetic beads on day 0 and transduced with TFP on day 1 using a bicistronic lentiviral vector expressing TFP along with eGFP using a 2A ribosomal skipping sequence.

[0280] Sustained TFP+ T cell expansion in the absence of re -stimulation can also be measured (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter® Multisizer III particle counter following stimulation with alphaCD3/alphaCD28 coated magnetic beads on day 0, and transduction with the indicated TFP on day 1

[0281] Animal models can also be used to measure a TFP T cell activity. For example, a xenograft model using human BCMA-specific TFP+ T cells to treat a cancer in immunodeficient mice is described in Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, after establishment of cancer, mice are randomized as to treatment groups. Different numbers of engineered T cells are coinjected at a 1: 1 ratio into NOD/SCID/Y-/- mice bearing cancer. The number of copies of each vector in spleen DNA from mice is evaluated at various times following T cell injection. Animals are assessed for cancer at weekly intervals. Peripheral blood tumor-associated antigen® cancer cell counts are measured in mice that are injected with alpha tumor-associated antigen-zeta TFP+ T cells or mock-transduced T cells. Survival curves for the groups are compared using the log -rank test. In addition, absolute peripheral blood CD4+ and CD8+ T cell counts 4 weeks following T cell injection in NOD/SCID/Y-/- mice can also be analyzed. Mice are injected with cancer cells and 3 weeks later are injected with T cells engineered to express TFP by a bicistronic lentiviral vector that encodes the TFP linked to eGFP. T cells are nonnalized to 45-50% input GFP+ T cells by mixing with mock-transduced cells prior to injection and confirmed by flow cytometry. Animals are assessed for cancer at l-week intervals. Survival curves for the TFP+ T cell groups are compared using the log-rank test.

[0282] Dose-dependent TFP treatment responses can be evaluated (see, e.g., Milone et ah, Molecular Therapy 17(8): 1453-1464 (2009)). For example, peripheral blood is obtained 35-70 days after establishing cancer in mice injected on day 21 with TFP T cells, an equivalent number of mock- transduced T cells, or no T cells. Mice from each group are randomly bled for determination of peripheral blood + cancer cell counts and then killed on days 35 and 49. The remaining animals are evaluated on days 57 and 70.

[0283] Assessment of cell proliferation and cytokine production has been previously described, e.g., at Milone et ah, Molecular Therapy 17(8): 1453-1464 (2009). Briefly, assessment of TFP-mediated proliferation is performed in microtiter plates by mixing washed T cells with cells expressing BCMA or CD32 and CD137 (KT32-BBL) for a final T celkcell expressing BCMA ratio of 2: l. Cells expressing BCMA cells are irradiated with gamma-radiation prior to use. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultures with KT32-BBL cells to serve as a positive control for stimulating T cell proliferation since these signals support long-term CD8+ T cell expansion ex vivo. T cells are enumerated in cultures using CountBright™ fluorescent beads (Invitrogen) and flow cytometry as described by the manufacturer. TFP+ T cells are identified by GFP expression using T cells that are engineered with eGFP-2A linked TFP -expressing lentiviral vectors. For TFP+ T cells not expressing GFP, the TFP+ T cells are detected with biotinylated recombinant BCMA protein and a secondary avidin-PE conjugate. CD4+ and CD8+ expression on T cells are also simultaneously detected with specific monoclonal antibodies (BD Biosciences). Cytokine measurements are performed on supernatants collected 24 hours following re -stimulation using the human TH1/TH2 cytokine cytometric bead array kit (BD Biosciences) according the manufacturer' s instructions. Fluorescence is assessed using a FACScalibur™ flow cytometer, and data is analyzed according to the manufacturer' s instructions.

[0284] Cytotoxicity can be assessed by a standard ⁵¹ Cr-release assay (see, e.g., Milone et ah, Molecular Therapy 17(8): 1453-1464 (2009)). For example, target cells are loaded with ⁵¹ Cr (as NaCr0 ₄ , New England Nuclear) at 37 ° C for 2 hours with frequent agitation, washed twice in complete RPMI and plated into microtiter plates. Effector T cells are mixed with target cells in the wells in complete RPMI at varying ratios of effector celktarget cell (E:T). Additional wells containing media only (spontaneous release, SR) or a 1% solution of Triton®-X 100 detergent (total release, TR) are also prepared. After 4 hours of incubation at 37 ° C, supernatant from each well is harvested. Released ⁵¹ Cr is then measured using a gamma particle counter (Packard Instrument Co., Waltham, Mass.). Each condition is performed in at least triplicate, and the percentage of lysis is calculated using the formula: % Lysis=(ER-SR)/(TR- SR), where ER represents the average ⁵¹ Cr released for each experimental condition.

[0285] Imaging technologies can be used to evaluate specific trafficking and proliferation of TFPs in tumor-bearing animal models. Such assays have been described, e.g., in Barrett et ah, Human Gene Therapy 22: 1575-1586 (2011). NOD/SCID/yc-/- (NSG) mice are injected intravenously (i.v.) with cancer cells followed 7 days later with TFP T cells 4 hours after electroporation with the TFP constructs. The T cells are stably transfected with a lentiviral construct to express firefly luciferase, and mice are imaged for bioluminescence. Alternatively, therapeutic efficacy and specificity of a single injection of TFP T cells in a cancer xenograft model can be measured as follows: NSG mice are injected with cancer cells transduced to stably express firefly luciferase, followed by a single tail -vein injection of T cells electroporated with BCMA TFP 7 days later. Animals are imaged at various time points post injection. For example, photon-density heat maps of firefly luciferase positive cancer in representative mice at day 5 (2 days before treatment) and day 8 (24 hours post TFP+ PBLs) can be generated.

[0286] Other assays, including those described in the Example section herein as well as those that are known in the art can also be used to evaluate the anti-TAA TFP constructs and TFP T cells disclosed herein.

Therapeutic Applications

Tumor Antigen-Associated Diseases or Disorders

[0287] While examples and embodiments have been provided herein, additional techniques and embodiments related to TAA -associated diseases and/or anti-TAA antibodies may be found in U.S.

Patent No. 9,217,040, filed January 13, 2013; U.S. Patent No. 9,758,586, filed November 30, 2011; International Publication No. WO 2012076066, filed June 17, 2011; Mato, A. & Porter, D. (2015) Blood 126(4), 478-485; Choi, M., et al. (2015) Clinical Lymphoma, Myeloma & Leukemia 15(S 1), S167-S169; Cui, B., et al. (2015) Cancer Research 73(12), 3649-3660; Yu, J., et al. (2015) Journal of Clinical Investigation 10(1172), 1-34; Borcherding, N., et al. (2014) Protein Cell 5(7), 496-502; Zhang, S., et al. (2012) The American Journal of Pathology 181(6), 1903-1910; Hudecek, M., et al. (2010) Blood 116(22), 4532-4541; and Deniger, D., et al. (2015) PLoS ONE 10(6), 1-19, which are entirely incorporated herein by reference.

[0288] In one aspect, the invention provides methods for treating a disease associated with a TAA, e.g., ROR1 or NKG2D ligand (NKG2DL) expression. In one aspect, the invention provides methods for treating a disease wherein part of the tumor is negative for NKG2DL and part of the tumor is positive for NKG2DL. For example, the TFP is useful for treating subjects that have undergone treatment for a disease associated with elevated expression of NKG2DL, wherein the subject that has undergone treatment for elevated levels of NKG2DL exhibits a disease associated with elevated levels of NKG2DL.

[0289] In one aspect, the invention pertains to a vector comprising a TAA-binding TFP operably linked to promoter for expression in mammalian T cells. In one aspect, the invention provides a recombinant T cell expressing the, e.g., NKG2D TFP for use in treating NKG2DL-expressing tumors, wherein the recombinant T cell expressing the NKG2D TFP is termed a NKG2D TFP T cell. In one aspect, the NKG2D TFP T cell is capable of contacting a tumor cell with at least one NKG2DL expressed on its surface such that the TFP T cell targets the tumor cell and growth of the tumor is inhibited.

Dual Specificity TFP T cells

[0290] Many patients treated with cancer therapeutics that are directed to one target on a tumor cell, e.g., BCMA, CD19, CD20, CD22, CD123, mesothelin, etc., become resistant over time as escape mechanisms such as alternate signaling pathways and feedback loops become activated. Dual specificity therapeutics attempt to address this by combining targets that often substitute for each other as escape routes.

Therapeutic T cell populations having TCRs specific to more than one tumor-associated antigen are promising combination therapeutics. In one embodiment, dual specificity TFP T cells comprise multiple blocking domains and corresponding multiple protease -cleavable linkers.

Tumor associated antigen targets for anti-TAA inducible TFP T cells

[0291] Exemplary tumor-associated antigens include, but are not limited to, oncofetal antigens (e.g., those expressed in fetal tissues and in cancerous somatic cells), oncoviral antigens (e.g., those encoded by tumorigenic transforming viruses), overexpressed/ accumulated antigens (e.g., those expressed by both normal and neoplastic tissue, with the level of expression highly elevated in neoplasia), cancer- testis antigens (e.g., those expressed only by cancer cells and adult reproductive tissues such as testis and placenta), lineage-restricted antigens (e.g., those expressed largely by a single cancer histotype), mutated antigens (e.g., those expressed by cancer as a result of genetic mutation or alteration in transcription), post-translationally altered antigens (e.g., those tumor-associated alterations in glycosylation, etc.), and idiotypic antigens (e.g., those from highly polymorphic genes where a tumor cell expresses a specific clonotype, e.g., as in B cell, T cell lymphoma/leukemia resulting from clonal aberrancies). Exemplary tumor-associated antigens include, but are not limited to, antigens of alpha- actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDK12, CDKN2A, CLPP, COA-l, CSNK1A1, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD, FNDC3B, FN1, GAS7, GPNMB, HAUS3, HSDL1, LDLR-fiicosyltransferase AS fusion protein, HLA-A2d, HLA-A1 ld, hsp70-2, MART2, MATN, ME1, MUM-lf, MUM-2, MUM-3, neo-PAP, Myosin class I, NFYC, OGT, OS-9, p53, pml-RARalpha fusion protein, PPP1R3B, PRDX5, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, TGF-betaRII, triosephosphate isomerase, BAGE-l, D393-CD20n, Cyclin-Al, GAGE-l, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GnTVf, HERV-K-MEL, KK-LC- 1, KM-HN-l, LAGE-l, LY6K, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE- A9, MAGE-A10, MAGE-A12 m, MAGE-C1, MAGE-C2, mucink, NA88-A, NY-ESO-l / LAGE-2, SAGE, Spl7, SSX-2, SSX-4, TAG-l, TAG-2, TRAG-3, TRP2-INT2g, X AGE- 1 b/GAGED2a, B7H4, DLL3, TROP-2, Nectin-4, tissue factor, LIV-1, CD48, cMET„ Gene / protein, CEA, gplOO / Pmell7, mammaglobin-A, Melan-A / MART-l, NY-BR-l, OA1, PAP, PSA, RAB38 / NY-MEL-l, TRP-l / gp75, TRP-2, tyrosinase, adipophilin, AIM-2, ALDH1A1, BCLX (L), BING-4, CALCA, CD45, CD274, CPSF, cyclin Dl, DKK1, ENAH (hMena), EpCAM, EphA3, EZH2, FGF5, glypican-3, HER-2/neu, HLA- DOB, Hepsin, IDOl, IGF2B3, ILl3Ralpha2, Intestinal carboxyl esterase, alpha-foetoprotein, Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP, mdm-2, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, p53, PAX5, PBF, PRAME, PSMA, RAGE-l, RGS5, RhoC, RNF43, RU2AS, secemin 1, SOX10, STEAP1, survivin, Telomerase, TPBG, VEGF, and WT1.

[0292] In one aspect, the invention provides methods for treating a disease associated with at least one tumor-associated antigen expression. In one aspect, the invention provides methods for treating a disease wherein part of the tumor is negative for the tumor associated antigen and part of the tumor is positive for the tumor associated antigen. For example, the antibody or TFP of the invention is useful for treating subjects that have undergone treatment for a disease associated with elevated expression of said tumor antigen, wherein the subject that has undergone treatment for elevated levels of the tumor associated antigen exhibits a disease associated with elevated levels of the tumor associated antigen.

[0293] In one aspect, the invention pertains to a vector comprising an anti-tumor-associated antigen antibody or TFP operably linked to promoter for expression in mammalian T cells. In one aspect, the invention provides a recombinant T cell expressing a tumor-associated antigen TFP for use in treating tumor-associated antigen-expressing tumors, wherein the recombinant T cell expressing the tumor- associated antigen TFP is termed a tumor-associated antigen TFP T cells. In one aspect, the tumor- associated antigen TFP T cell of the present disclosure is capable of contacting a tumor cell with at least one tumor-associated antigen TFP of the present disclosure expressed on its surface such that the TFP T cell targets the tumor cell and growth of the tumor is inhibited.

[0294] In one aspect, the invention pertains to a method of inhibiting growth of a tumor-associated antigen-expressing tumor cell, comprising contacting the tumor cell with a tumor-associated antigen antibody or TFP T cell of the present disclosure such that the TFP T is activated in response to the antigen and targets the cancer cell, wherein the growth of the tumor is inhibited.

[0295] In one aspect, the present disclosure pertains to a method of treating cancer in a subject. The method comprises administering to the subject a tumor-associated antigen antibody, bispecific antibody, or TFP T cell of the present disclosure such that the cancer is treated in the subject. An example of a cancer that is treatable by the tumor-associated antigen TFP T cell of the present disclosure is a cancer associated with expression of tumor-associated antigen. In one aspect, the cancer is a myeloma. In one aspect, the cancer is a lymphoma. In one aspect, the cancer is colon cancer.

[0296] In some embodiments, tumor-associated antigen antibodies or TFP therapy can be used in combination with one or more additional therapies. In some instances, such additional therapies comprise a chemotherapeutic agent, e.g., cyclophosphamide. In some instances, such additional therapies comprise surgical resection or radiation treatment.

[0297] In one aspect, disclosed herein is a method of cellular therapy wherein T cells are genetically modified to express a TFP and the TFP-expressing T cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Unlike antibody therapies, TFP-expressing T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control. In various aspects, the T cells administered to the patient, or their progeny, persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty -one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of the T cell to the patient.

[0298] In some instances, disclosed herein is a type of cellular therapy where T cells are modified, e.g., by in vitro transcribed RNA, to transiently express a TFP and the TFP-expressing T cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Thus, in various aspects, the T cells administered to the patient, is present for less than one month, e.g., three weeks, two weeks, or one week, after administration of the T cell to the patient.

[0299] Without wishing to be bound by any particular theory, the anti -tumor immunity response elicited by the TFP-expressing T cells may be an active or a passive immune response, or alternatively may be due to a direct vs indirect immune response. In one aspect, the TFP transduced T cells exhibit specific proinflammatory cytokine secretion and potent cytolytic activity in response to human cancer cells expressing the tumor-associated antigen, resist soluble tumor-associated antigen inhibition, mediate bystander killing and/or mediate regression of an established human tumor. For example, antigen-less tumor cells within a heterogeneous field of tumor-associated antigen-expressing tumor may be susceptible to indirect destruction by tumor-associated antigen-redirected T cells that has previously reacted against adjacent antigen-positive cancer cells.

[0300] In one aspect, the human TFP-modified T cells of the present disclosure may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one aspect, the mammal is a human.

[0301] With respect to ex vivo immunization, at least one of the following occurs in vitro prior to administering the cell into a mammal: i) expansion of the cells, ii) introducing a nucleic acid encoding a TFP to the cells or iii) cryopreservation of the cells.

[0302] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a TFP disclosed herein. The TFP-modified cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the TFP-modified cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.

[0303] The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described, e.g., in U.S. Patent No. 5,199,942, incorporated herein by reference, can be applied to the cells of the present disclosure. Other suitable methods are known in the art; therefore, the present disclosure is not limited to any particular method of ex vivo expansion of the cells. Briefly, ex vivo culture and expansion of T cells comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo. In addition to the cellular growth factors described in U.S. Patent No. 5,199,942, other factors such as flt3-L, IL-l, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.

[0304] In addition to using a cell -based vaccine in terms of ex vivo immunization, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.

[0305] Generally, the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, the TFP- modified T cells of the present disclosure are used in the treatment of diseases, disorders and conditions associated with expression of tumor-associated antigens. In certain aspects, the cells of the present disclosure are used in the treatment of patients at risk for developing diseases, disorders and conditions associated with expression of tumor-associated antigens. Thus, the present disclosure provides methods for the treatment or prevention of diseases, disorders and conditions associated with expression of tumor- associated antigens comprising administering to a subject in need thereof, a therapeutically effective amount of the TFP -modified T cells of the present disclosure.

[0306] In one aspect, the antibodies or TFP T cells disclosed herein may be used to treat a proliferative disease such as a cancer or malignancy or is a precancerous condition. In one aspect, the cancer is a myeloma. In one aspect, the cancer is a lymphoma. In one aspect, the cancer is a colon cancer. Further, a disease associated with tumor-associated antigen expression includes, but is not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing tumor-associated antigens. Non-cancer related indications associated with expression of tumor-associated antigens vary depending on the antigen, but are not limited to, e.g., infectious disease, autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma) and transplantation.

[0307] The antibodies or TFP-modified T cells of the present disclosure may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-7, IL-12, IL-15 or other cytokines or cell populations.

[0308] The present disclosure also provides methods for inhibiting the proliferation or reducing a tumor- associated antigen-expressing cell population, the methods comprising contacting a population of cells comprising a tumor-associated antigen-expressing cell with an anti -tumor-associated antigen TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell. In a specific aspect, the present disclosure provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing tumor-associated antigen, the methods comprising contacting the tumor- associated antigen-expressing cancer cell population with an anti-tumor-associated antigen antibody or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell. In one aspect, the present disclosure provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing tumor-associated antigen, the methods comprising contacting the tumor- associated antigen-expressing cancer cell population with an anti-tumor-associated antigen antibody or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell. In certain aspects, the anti-tumor-associated antigen antibody or TFP T cell of the present disclosure reduces the quantity, number, amount or percentage of cells and/or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a subject with or animal model for multiple myeloma or another cancer associated with tumor-associated antigen expressing cells relative to a negative control. In one aspect, the subject is a human.

[0309] The present disclosure also provides methods for preventing, treating and/or managing a disease associated with tumor-associated antigen-expressing cells (e.g., a cancer expressing tumor-associated antigen), the methods comprising administering to a subject in need an anti-tumor-associated antigen antibody or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell. In one aspect, the subject is a human. Non-limiting examples of disorders associated with tumor- associated antigen-expressing cells include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma) and cancers (such as hematological cancers or atypical cancers expressing tumor-associated antigen).

[0310] The present disclosure also provides methods for preventing, treating and/or managing a disease associated with tumor-associated antigen-expressing cells, the methods comprising administering to a subject in need an anti-tumor-associated antigen antibody or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell. In one aspect, the subject is a human.

[0311] The present disclosure provides methods for preventing relapse of cancer associated with tumor- associated antigen-expressing cells, the methods comprising administering to a subject in need thereof an anti-tumor-associated antigen antibody and/or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell. In one aspect, the methods comprise administering to the subject in need thereof an effective amount of an anti -tumor-associated antigen antibody or TFP T cell described herein that binds to the tumor-associated antigen-expressing cell in combination with an effective amount of another therapy.

Combination Therapies

[0312] An inducible TFP-expressing cell described herein may be used in combination with other known agents and therapies. Administered "in combination", as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject' s affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons.

In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

Other Combinations

[0313] In some embodiments, the "at least one additional therapeutic agent" includes a TFP-expressing cell. Also provided are T cells that express multiple TFPs, which bind to the same or different target antigens, or same or different epitopes on the same target antigen. Also provided are populations of T cells in which a first subset of T cells expresses a first TFP and a second subset of T cells express a second TFP.

[0314] A TFP-expressing cell described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the TFP-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.

[0315] In further aspects, a TFP-expressing cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cyclophosphamide, fludarabine, cyclosporin, tacrolimus (fujimycin), rapamycin, mycophenolic acid, steroids, romidepsin (also known as FR901228), cytokines, and irradiation peptide vaccine, such as that described in, e.g., Izumoto et al.

2008 J Neurosurg 108:963-971.

[0316] In one embodiment, the subject can be administered an agent which reduces or ameliorates a side effect associated with the administration of a TFP-expressing cell. Side effects associated with the administration of a TFP-expressing cell include, but are not limited to, cytokine release syndrome (CRS), and hemophagocytic lymphohistiocytosis (HLH), also termed Macrophage Activation Syndrome (MAS). Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like.

Accordingly, the methods described herein can comprise administering a TFP-expressing cell described herein to a subject and further administering an agent to manage elevated levels of a soluble factor resulting from treatment with a TFP-expressing cell. In one embodiment, the soluble factor elevated in the subject is one or more of IFN-g, TNFc IL-2, IL-6, and IL-8. Therefore, an agent administered to treat this side effect can be an agent that neutralizes one or more of these soluble factors. Such agents include, but are not limited to a steroid, an inhibitor of TNFa, and an inhibitor of IL-6. An example of a TNFa inhibitor is etanercept (marketed under the name ENBREL®). An example of an IL-6 inhibitor is tocilizumab (marketed under the name ACTEMRA®).

[0317] In one embodiment, the subject can be administered an agent which enhances the activity of a TFP-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., Programmed Death 1 (PD1), can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. Inhibition of an inhibitory molecule, e.g., by inhibition at the DNA, RNA or protein level, can optimize a TFP-expressing cell performance. In embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, can be used to inhibit expression of an inhibitory molecule in the TFP-expressing cell. In an embodiment, the inhibitor is a shRNA. In an embodiment, the inhibitory molecule is inhibited within a TFP-expressing cell. In these embodiments, a dsRNA molecule that inhibits expression of the inhibitory molecule is linked to the nucleic acid that encodes a component, e.g., all of the components, of the TFP. In one embodiment, the inhibitor of an inhibitory signal can be, e.g., an antibody or antibody fragment that binds to an inhibitory molecule. For example, the agent can be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX-101, and marketed as YERVOY®); Bristol-Myers Squibb; tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP -675, 206)). In an embodiment, the agent is an antibody or antibody fragment that binds to T cell immunoglobulin and mucin-domain containing-3 (TIM3). In an embodiment, the agent is an antibody or antibody fragment that binds to Lymphocyte -activation gene 3 (LAG3).

[0318] In some embodiments, an agent suitable for use in combination with the TFP T cells disclosed herein is an agent that modulates myeloid suppressor cells, e.g., CCR2 antibodies. Other therapeutics, e.g, nanoparticle therapeutics, are known in the art.

[0319] In some embodiments, the agent which enhances the activity of a TFP-expressing cell can be, e.g., a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule, or fragment thereof, and the second domain is a polypeptide that is associated with a positive signal, e.g., a polypeptide comprising an intracellular signaling domain as described herein. In some embodiments, the polypeptide that is associated with a positive signal can include a costimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27 and/or ICOS, and/or a primary signaling domain, e.g., of CD3 zeta, e.g., described herein. In one embodiment, the fusion protein is expressed by the same cell that expressed the TFP. In another embodiment, the fusion protein is expressed by a cell, e.g., a T cell that does not express an anti-tumor-associated antigen TFP. Pharmaceutical Compositions

[0320] Pharmaceutical compositions of the present disclosure may comprise a TFP-expressing cell, e.g., a plurality of TFP-expressing cells, as described herein, in combination with one or more

pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure are in one aspect formulated for intravenous administration.

[0321] Pharmaceutical compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient ' s disease, although appropriate dosages may be determined by clinical trials.

[0322] In one embodiment, the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus. In one embodiment, the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.

[0323] When "an immunologically effective amount," "an anti-tumor effective amount," "a tumor- inhibiting effective amount," or "therapeutic amount" is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 104 to 109 cells/kg body weight, in some instances 10 ⁵ to 10 ⁶ cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et ah, New Eng. J. of Med. 319: 1676, 1988).

[0324] In certain aspects, it may be desired to administer activated T cells to a subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom according to the present disclosure, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain aspects, T cells can be activated from blood draws of from 10 cc to 400 cc. In certain aspects, T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

[0325] The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one aspect, the T cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection. In one aspect, the T cell compositions of the present disclosure are administered by i.v. injection. The compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.

[0326] In a particular exemplary aspect, subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g., T cells. These T cell isolates may be expanded by methods known in the art and treated such that one or more TFP constructs of the present disclosure may be introduced, thereby creating a TFP -expressing T cell of the present disclosure. Subjects in need thereof may subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain aspects, following or concurrent with the transplant, subjects receive an infusion of the expanded TFP T cells of the present disclosure. In an additional aspect, expanded cells are administered before or following surgery.

[0327] The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices. The dose for alemtuzumab (CAMPATH®), for example, will generally be in the range 1 to about 100 mg for an adult patient, usually administered daily for a period between 1 and 30 days. The preferred daily dose is 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used (described in U.S.

Patent No. 6,120,766).

[0328] In one embodiment, the TFP is introduced into T cells, e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of TFP T cells of the present disclosure, and one or more subsequent administrations of the TFP T cells of the present disclosure, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, more than one administration of the TFP T cells of the present disclosure are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the TFP T cells of the present disclosure are administered per week. In one embodiment, the subject (e.g., human subject) receives more than one administration of the TFP T cells per week (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no TFP T cells administrations, and then one or more additional administration of the TFP T cells (e.g., more than one administration of the TFP T cells per week) is administered to the subject. In another embodiment, the subject (e.g., human subject) receives more than one cycle of TFP T cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the TFP T cells are administered every other day for 3 administrations per week. In one embodiment, the TFP T cells of the present disclosure are administered for at least two, three, four, five, six, seven, eight or more weeks.

[0329] In one aspect, tumor-associated antigen TFP T cells are generated using lentiviral viral vectors, such as lentivirus. TFP T cells generated that way will have stable TFP expression.

[0330] In one aspect, TFP T cells transiently express TFP vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15 days after transduction. Transient expression of TFPs can be effected by RNA TFP vector delivery. In one aspect, the TFP RNA is transduced into the T cell by electroporation.

[0331] A potential issue that can arise in patients being treated using transiently expressing TFP T cells (particularly with murine scFv bearing TFP T cells) is anaphylaxis after multiple treatments.

[0332] Without being bound by this theory, it is believed that such an anaphylactic response might be caused by a patient developing humoral anti-TFP response, i.e., anti-TFP antibodies having an anti-IgE isotype. It is thought that a patient' s antibody producing cells undergo a class switch from IgG isotype (that does not cause anaphylaxis) to IgE isotype when there is a ten- to fourteen-day break in exposure to antigen.

[0333] If a patient is at high risk of generating an anti-TFP antibody response during the course of transient TFP therapy (such as those generated by RNA transductions), TFP T cell infusion breaks should not last more than ten to fourteen days.

EXAMPLES

[0334] The present disclosure is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the present disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present disclosure and practice the claimed methods. The following working examples specifically point out various aspects of the present disclosure and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1: TFP Constructs

[0335] Anti-TAA TFP constructs are engineered by cloning one or more anti-TAA scFv DNA fragment linked to a CD3 or TCR DNA fragment by either a DNA sequence encoding a short linker (SL):

AAAGGGGSGGGGSGGGGSLE (SEQ ID NO: 188) or a long linker (LL):

AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE (SEQ ID NO: 189) into, e g., r510 vector ((System Biosciences (SBI)) at Xbal and EcoRl sites. CAR constructs are generated by cloning synthesized DNA encoding an anti-TAA antibody, partial CD28 extracellular domain, CD28 transmembrane domain,

CD28 intracellular domain and CD3 zeta into, e.g., a p510 vector at Xbal and EcoRl sites. CD3 e TFP constructs disclosed herein comprise the sequence set forth in SEQ ID NO: 191, which has an N-terminal truncation in reference to the full sequence (SEQ ID NO: 192).

[0336] In one embodiment, an anti-tumor-associated antigen CAR construct is generated as a comparator. A p5 lO_antitumor-associated antigen_28 z CAR is generated by cloning synthesized DNA encoding anti-tumor-associated antigen, partial CD28 extracellular domain, CD28 transmembrane domain, CD28 intracellular domain and CD3 zeta into p510 vector at Xbal and EcoRl sites.

Protease- cleavable anti-HSA/anti-MSLN Fusion Constructs

[0337] An anti-HSA sdAb (SEQ ID NO:42) was genetically fused to anti-MSLN sdAb SD1 (SEQ ID NO:39) via a cleavable linker. See, e.g., FIG. 6, which shows inducible TFP construct sequences in the format 5 ' -anti-HSA sdAb— protease-cleavable linker— anti-MSLN sdAb binder-3 ' ) with a C-terminal 6His tag for purification and detection purposes. Fusion proteins were expressed in E. coli and purified to homogeneity by Ni-NTA affinity chromatography. Fusion proteins were desalted and stored and in lxPBS buffer, pH 7.4.

Example 2: Antibody Sequences

Generation of Antibody Sequences

[0338] Provided are antibody polypeptides, fragments thereof, single domain antibodies, Fab fragments, and other antigen binding proteins that are capable of specifically binding to the human polypeptide (s), and fragments or domains thereof. In some embodiments, the antigen is a tumor-associated antigen (TAA). Anti-TAA antibodies or fragments thereof can be generated using diverse technologies (see, e.g., (Nicholson et al, 1997). Where murine anti-TAA antibodies are used as a starting material, humanization of murine anti-TAA antibodies is desired for the clinical setting, where the mouse-specific residues may induce a human-anti-mouse antigen (HAMA) response in subjects who receive T cell receptor (TCR) fusion protein (TFP) treatment, i.e., treatment with T cells transduced with the anti-TAA TFP construct. Humanization is accomplished by grafting CDR regions from murine anti-TAA antibody onto appropriate human germline acceptor frameworks, optionally including other modifications to CDR and/or framework regions. As provided herein, antibody and antibody fragment residue numbering follows Kabat (Kabat E. A. et al, 1991; Chothia et al, 1987).

Generation of scFvs

[0339] Human or humanized anti-TAA IgGs are used to generate scFv sequences for TFP constructs. DNA sequences coding for human or humanized VF and VH domains are obtained, and the codons for the constructs are, optionally, optimized for expression in cells from Homo sapiens. The order in which the VF and VH domains appear in the scFv is varied (i.e., VF-VH, or VH-VF orientation), and three copies of the "G ₄ S" or "G ₄ S" subunit (G ₄ S) ₃ connect the variable domains to create the scFv domain. Anti-TAA scFv plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK-293 or other suitable human or mammalian cell lines and purified. Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of TAA- expressing cells.

Source of TCR Subunits

[0340] Subunits of the human T Cell Receptor (TCR) complex all contain an extracellular domain, a transmembrane domain, and an intracellular domain. A human TCR complex contains the CD3 -epsilon polypeptide, the CD3-gamma polypeptide, the CD3-delta polypeptide, the CD3-zeta polypeptide, the TCR alpha chain polypeptide and the TCR beta chain polypeptide. The human CD3-epsilon polypeptide canonical sequence is UniProt Accession No. P07766. The human CD3-gamma polypeptide canonical sequence is UniProt Accession No. P09693. The human CD3-delta polypeptide canonical sequence is UniProt Accession No. P043234. The human CD3-zeta polypeptide canonical sequence is UniProt Accession No. P20963. The human TCR alpha chain canonical sequence is UniProt Accession No. Q6ISU1. The human TCR beta chain C region canonical sequence is UniProt Accession No. P01850, a human TCR beta chain V region sequence is P04435.

[0341] The human CD3 -epsilon polypeptide canonical sequence is:

MQSGTHWRVFGFCFFSVGVWGQDGNEEMGGITQTPYKVSISGTTVIFTCPQYPGSEIFWQ HND KNIGGDEDDKNIGSDEDHFSFKEFSEFEQSGYYVCYPRGSKPEDANFYFYFRARVCENCM EMD VMSVAT]VrVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNP DYEP IRKGQRDLYSGLNQRRI (SEQ ID NO: 193). In one embodiment, the human CD3-epsilon fragment used in the TFPs is

DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHL SLKEFS ELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLV YYWS KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 194)

[0342] The human CD3 -gamma polypeptide canonical sequence is:

MEOGKGT .AVT JT .ATTT J QGTT AOSTKGNHT ,VK VYPY OEPGSVT J TCDAEAKNTTWFKDGKMTGF LTEDKKKWNLGSNAKDPRGMY QCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIV SIFV LAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 195). In one embodiment, the human CD3-gamma fragment used in the TFPs is:

QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAK DPRGM YQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQ SRASD KQTLLPNDQLY QPLKDREDDQY SHLQGNQLRRN (SEQ ID NO: 196).

[0343] The human CD3 -delta polypeptide canonical sequence is:

MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDL GKRILDP RGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGHVTDVIATLLLALGVFCFAGH ETGR LSGAADTQALLRNDQVY QPLRDRDDAQY SHLGGNWARNK (SEQ ID NO: 197). In one embodiment, the human CD3 -delta fragment used in the TFPs is:

FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKD KESTVQV HYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQV YQPL RDRDDAQY SHLGGNWARNK (SEQ ID NO: 198)

[0344] The human CD3-zeta polypeptide canonical sequence is:

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSAD APAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEI GM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 199). In one embodiment, the human CD3-zeta fragment used in the TFPs is:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLY NE LQKDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQ ALPPR (SEQ ID NO:300)

[0345] The human TCR alpha chain canonical sequence is:

MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSP IWFS AGNGSALDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMH LSGE ASTARTCPQEPLRGTPGGALWLGVLRLLLFKLLLFDLLLTCSCLCDPAGPLPSPATTTRL RALGS HRLHPATETGGRE ATS SPRPQPRDRRW GDTPPGRKPGSP VWGEGS YLS S YPT CP AQ AW C SRS AL RAPSSSLGAFFAGDLPPPLQAGAA (SEQ ID NO:20l).

[0346] The human TCR alpha chain C region canonical sequence is:

PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKS NSAV AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL LLKVAGF NLLMTLRLW S S (SEQ ID NO: 202)

[0347] The human TCR alpha chain V region CTL-L17 canonical sequence is:

MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFD YFLWY KKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAAKGAG TASKLT F GTGTRLQ VTL (SEQ ID NO:203).

[0348] The human TCR beta chain C region canonical sequence is:

EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDP QPLK EQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAE AWG RADCGFTS V SYQQGVLS ATILYEILLGKATLYAVLV S ALVLMAMVKRKDF (SEQ ID NO:204). [0349] The human TCR beta chain V region CTL-L17 canonical sequence is:

MGTSLLCWMALCLLGADHADTGVSQNPRHNITKRGQNVTFRCDPISEHNRLYWYRQTLGQ GPE FLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLAGLNQPQH FGDGT RLSIL (SEQ ID NO:205).

[0350] The human TCR beta chain V region YT35 canonical sequence is:

MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMR GLE LLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSFSTCSANYG YTFGSG TRLTVV (SEQ ID NO: 206).

Generation of TFPs from TCR Domains and scFvs, Fab fragments, or sdAbs

[0351] Anti-TAA scFvs are recombinantly linked to a) CD3-epsilon or other TCR subunits, and b) a blocking domain, such as HSA, using a linker sequence, such as G4S, (G4S)2 (G4S)3 or (G4S)4. Various linkers and antibody (e.g., scFv or sdAb) configurations are used. TCR alpha and TCR beta chains are used for generation of TFPs either as full-length polypeptides or as only their constant domains (e.g., in the preparation for a construct suitable for an allogeneic immune cell product) . Any variable sequence of TCR alpha and TCR beta chains is suitable for making TFPs.

TFP Expression Vectors

[0352] Expression vectors are provided that include: a promoter (Cytomegalovirus (CMV) enhancer- promoter), a signal sequence to enable secretion, a polyadenylation signal and transcription terminator (Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes (e.g., SV40 origin and ColEl or others known in the art) and elements to allow selection (ampicillin resistance gene and zeocin marker).

[0353] Preferably, the TFP -encoding nucleic acid construct or constructs is/are cloned into one or more lentiviral expression vectors and expression validated based on the quantity and quality of the effector T cell response of transduced T cells in response to TAA+ target cells. Effector T cell responses include, but are not limited to, cellular expansion, proliferation, doubling, cytokine production and target cell lysis or cytolytic activity (i.e., degranulation).

[0354] The TFP lentiviral transfer vectors are used to produce the genomic material packaged into the VSVg pseudotyped lentiviral particles. Lentiviral transfer vector DNA is mixed with the three packaging components of VSVg, gag/pol and rev in combination with Lipofectamine® reagent to transfect them together into 293 cells. After 24 and 48 hours, the media is collected, filtered and concentrated by ultracentrifugation. The resulting viral preparation is stored at -80° C. The number of transducing units is determined by titration on SupTl cells (T cell lymphoblastic lymphoma, (ATCC® CRL-1942™).

Redirected dual specificity TFP T cells are produced by activating fresh naive T cells with anti-CD3x anti-CD28 beads for 24 hrs and then adding the appropriate number of transducing units to obtain the desired percentage of transduced T cells. These modified T cells are allowed to expand until they become rested and come down in size at which point they are cryopreserved for later analysis. The cell numbers and sizes are measured using a Coulter Counter® Multisizer™ 3 (Beckman Coulter). Before

cryopreserving, percentage of cells transduced (expressing TFP.BCMA on the cell surface) and their relative fluorescence intensity of that expression are determined by flow cytometric analysis. From the histogram plots, the relative expression levels of the TFPs are examined by comparing percentage transduced with their relative fluorescent intensity.

[0355] In some embodiments, the vector is an adenoviral vector. In some embodiments, the vector is a circular RNA or a circular RNA transfer vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, multiple TFPs are introduced by T cell transduction with multiple viral vectors. Evaluating Cytolytic Activity. Proliferation Capabilities and Cytokine Secretion of Humanized TFP Redirected T Cells

[0356] The functional abilities of TFP.TAA T cells to produce cell-surface-expressed TFPs, and to kill target tumor cells, proliferate and secrete cytokines are determined using assays known in the art.

Cleavage of the protease-cleavable linker(s) tethering the blocking domain to the TFP T cell can be tested, for example, in vitro by proteolytic cleavage followed by western blot analysis of cleavage fragments.

[0357] Human PBMCs (e.g., blood from a normal apheresed donor whose naive T cells are obtained by negative selection for T cells, CD4+ and CD8+ lymphocytes) are treated with human interleukin-2 (IL-2) then activated with anti-CD3x anti-CD28 beads, e.g., in 10% RPMI at 37 ° C, 5% C0 ₂ prior to transduction with the TFP -encoding lentiviral vectors. Flow cytometry assays are utilized to confirm cell surface presence of a TFP, such as by an anti-FLAG antibody or an anti-murine variable domain antibody. Cytokine (e.g., IFN-g) production is measured using ELISA or other assays.

Example 3 : Human TFP T Cell Treatment in an In Vivo Solid Tumor Xenograft Mouse Model

[0358] The efficacy of human inducible TFP T cells can also be tested in immune compromised mouse models bearing subcutaneous solid tumors derived from human TAA-expressing human cell lines.

Tumor shrinkage in response to human inducible TFP T cell treatment can be either assessed by caliper measurement of tumor size, or by following the intensity of a GFP fluorescence signal emitted by GFP- expressing tumor cells.

[0359] Primary human solid tumor cells can be grown in immune-compromised mice without having to culture the cells in vitro. Exemplary solid cancer cells include solid tumor cell lines, such as provided in The Cancer Genome Atlas (TCGA) and/or the Broad Cancer Cell Line Encyclopedia (CCLE, see Barretina et ah, Nature 483:603 (2012)). Exemplary solid cancer cells include primary tumor cells isolated from mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney, endometrial, or stomach cancer. In some embodiments, the cancer to be treated is selected from the group consisting of mesotheliomas, papillary serous ovarian adenocarcinomas, clear cell ovarian carcinomas, mixed Mullerian ovarian carcinomas, endometroid mucinous ovarian carcinomas, pancreatic adenocarcinomas, ductal pancreatic adenocarcinomas, uterine serous carcinomas, lung adenocarcinomas, extrahepatic bile duct carcinomas, gastric adenocarcinomas, esophageal adenocarcinomas, colorectal adenocarcinomas and breast adenocarcinomas. These mice can be used to test the efficacy of anti-TAA TFP T cells in the human tumor xenograft models (see, e.g., Morton et ah, Nat. Procol. 2:247 (2007)). Following an implant or injection of 1 x 10 ⁶ - 1 x 10 ⁷ primary cells (collagenase -treated bulk tumor suspensions in EC matrix material) or tumor fragments (primary tumor fragments in EC matrix material) subcutaneously, tumors are allowed to grow to 200-500 mm ³ prior to initiation of treatment.

Example 4: Preparation of T Cells Transduced with Inducible TFPs

Lentiviral production

[0360] In one embodiment, the inducible TFP is encoded by a lentivirus. Lentivirus encoding the appropriate constructs are prepared as follows. 5 x 10 ⁶ HEK-293FT cells are seeded into a 100 mm dish and allowed to reach 70-90% confluency overnight. 2.5 pg of the indicated DNA plasmids and 20 pL Lentivirus Packaging Mix (ALSTEM, cat# VP100) are diluted in 0.5 mL DMEM or Opti-MEM® I Medium without serum and mixed gently. In a separate tube, 30 pL of NanoFect® transfection reagent (ALSTEM, cat# NF100) is diluted in 0.5 mL DMEM or Opti-MEM I Medium without serum and mixed gently. The NanoFect/DMEM and DNA/DMEM solutions are then mixed together and vortexed for 10- 15 seconds prior to incubation of the DMEM-plasmid-NanoFect mixture at room temperature for 15 minutes. The complete transfection complex from the previous step is added dropwise to the plate of cells and rocked to disperse the transfection complex evenly in the plate. The plate is then incubated overnight at 37 ° C in a humidified 5% C0 ₂ incubator. The following day, the supernatant is replaced with 10 mL fresh medium and supplemented with 20 pL of ViralBoost™ (500x, ALSTEM, cat# VB100). The plates are then incubated at 37 ° C for an additional 24 hours. The lentivirus containing supernatant is then collected into a 50 mL sterile, capped conical centrifuge tube and put on ice. After centrifugation at 3000 rpm for 15 minutes at 4° C, the cleared supernatant is filtered with a low-protein binding 0.45 pm sterile filter and virus is subsequently isolated by ultracentrifugation at 25,000 rpm (Beckmann, L8-70M) for 1.5 hours, at 4° C. The pellet is removed and re-suspended in DMEM medium and lentivirus

concentrations/titers are established by quantitative RT-PCR using the Lenti-X™ qRT-PCR Titration kit (Clontech®; catalog number 631235). Any residual plasmid DNA is removed by treatment with DNasel. The virus stock preparation is either used for infection immediately or aliquoted and stored at -80 ° C for future use.

[0361] Lentivirus titers are established by transducing cells (e.g., Jurkat cells) with different amount of virus preparation. The DNA is then isolated from the transduced Jurkat cells 24 hours after transduction. The virus titer is determined, e.g., by quantitative real-time PCR, with in-house designed primers/probe specific for Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) as well as for an internal quantitation control.

T cell isolation

[0362] Peripheral Blood Mononuclear Cells (PBMCs) are prepared from either whole blood or buffy coat. Whole blood is collected in 10 mL Heparin vacutainers and either processed immediately or stored overnight at 4 ° C. Approximately 10 mL of whole anti -coagulated blood is mixed with sterile phosphate buffered saline (PBS) buffer for a total volume of 20 mL in a 50 mL conical centrifuge tube (PBS, pH 7.4, without Ca ²⁺ /Mg ²⁺ ). 20 mL of this blood/PBS mixture is then gently overlaid onto the surface of 15 mL of Ficoll-Paque® PLUS (GE Healthcare, 17-1440-03) prior to centrifugation at 400g for 30-40 min at room temperature with no brake application.

[0363] Buffy coat is purchased, e.g., from Research Blood Components (Boston, MA). LeucoSep™ tubes (Greiner bio-one) are prepared by adding 15 mL Ficoll-Paque and centrifuged at lOOOg for 1 minute. Buffy coat is diluted 1 :3 in PBS (pH 7.4, without Ca ²⁺ or Mg ²⁺ ). The diluted buffy coat is transferred to LeucoSep tube and centrifuged at lOOOg for 15 minutes with no brake application. The layer of cells containing PBMCs, seen at the diluted plasma/Ficoll interface, is removed carefully to minimize contamination by Ficoll®. Residual Ficoll, platelets, and plasma proteins are then removed by washing the PBMCs three times with 40 mL of PBS by centrifugation at 200g for 10 minutes at room temperature. The cells are then counted with a hemocytometer. CD4+ and CD8+ T cells are then frozen down in freezing medium (90% FBS+l0% DMS0 at a concentration of 30-50 x 10 ⁶ cells per vial.

T cell activation

[0364] PBMCs prepared from either whole blood or buffy coat are stimulated with anti-human CD28 and CD3 antibody -conjugated magnetic beads for 24 hours prior to viral transduction. Freshly isolated PBMCs are washed once in CAR T medium (AIM V-AlbuMAX BSA, Life Technologies), with 5% AB serum and 1.25 pg/mL amphotericin B (Gemini Bioproducts), 100 U/mL penicillin, and 100 pg/mL streptomycin) without huIL-2, before being re-suspended at a final concentration of 1 x 10 ⁶ cells/mL in CAR T medium with 300 IU/mL human IL-2, IL-7, or IL-15 (from a lOOOx stock; Invitrogen).

[0365] Alternatively, frozen CD4+/CD8+ T cells are thawed in pre-warmed DMEM + 10 % FBS, spun down, and then resuspended in complete T cell expansion medium supplemented with 300 IU/mL huIL2 (Thermo Fisher®) at a final concentration of 1 x 10 ⁶ cells/mL. Prior to being used to activate T cells, anti human CD28 and anti-human CD3 antibody-conjugated magnetic beads (Dynabeads®, Thermo Fisher) are washed three times with sterile 1 x PBS (pH7.4), using a magnetic rack to isolate beads from the solution. The T cells are then mixed with the beads at a 1 : 1 ratio, by transferring 25 pL (lxlO ⁶ beads) of beads to 1 mL of T cell suspension. The beads/cells mixture is then dispensed to single wells of a non-TC treated l2-well plate, and incubated at 37 ° C with 5 % C0 ₂ for 24 hrs.

[0366] Prior to activation, anti-human CD28 and CD3 antibody-conjugated magnetic beads (available from, e.g., Invitrogen, Life Technologies) are washed three times with 1 mL of sterile lx PBS (pH 7.4), using a magnetic rack to isolate beads from the solution, before re -suspension in CAR T medium, with 300 IU/mL human IL-2, to a final concentration of 4 x 10 ⁷ beads/mL. PBMC and beads are then mixed at a 1 : 1 bead-to-cell ratio, by transferring 25 pL (lxlO ⁶ beads) of beads to 1 mL of PBMC. The desired number of aliquots are then dispensed to single wells of a l2-well low-attachment or non-treated cell culture plate, and incubated at 37 ° C, with 5% C0 ₂ , for 24 hours before viral transduction.

T cell transduction and expansion

[0367] Following activation of PBMCs, cells are incubated for 24 hours at 37 ° C, 5% C0 ₂ . Lentivirus is thawed on ice and then added to activated T cells at indicated MOI in the presence of 10 pg/ml Polybrene (Sigma). Cells are spinoculated with the lentivirus at 200 g for 100 minutes at room temperature. The transduced T cells are incubated for an additional 24 hr before an additional lentivirus transduction. After the 2nd round of lentivirus transduction, the T cells are expanded in T cell expansion medium supplemented with 300 IU/mL of hIL-2 and sub-cultured every other day at 5 x 10 ⁵ cells/mL.

[0368] In some instances, activated PBMCs are electroporated with in vitro transcribed (IVT) mRNA. Human PBMCs are stimulated with Dynabeads® (Thermo Fisher®) at l-to-l ratio for 3 days in the presence of 300 IU/ml recombinant human IL-2 (R&D System). The beads are removed before electroporation. The cells are washed and re-suspended in OPTI-MEM® medium (Thermo Fisher) or AimV® medium (Invitrogen) in 5% hAB serum (Gemini Bio-Products) and 1% antibiotics at the concentration of 2.5 x 10 ⁷ cells/mL. 200 pL of the cell suspension (5 x 10 ⁶ cells) are transferred to the 2 mm gap Electroporation Cuvettes Plus™ (Harvard Apparatus BTX) and prechilled on ice. 10 pg of IVT TFP mRNA is added to the cell suspension. The mRNA/cell mixture is then electroporated at 200 V for 20 milliseconds using ECM® 830 Electro Square Wave Porator (BTX® Harvard Apparatus).

Immediately after the electroporation, the cells are transferred to fresh cell culture medium (AIM V AlbuMAX® (BSA) serum free medium + 5% human AB serum + 300 IU/ml IL-2) and incubated at 37° C.

Example 5. Protease Inducible TFP T cells using MMP-Cleavable Blocking Domains

[0369] In this Example, linkers for an inducible TFP T cell comprising a biologically active polypeptide fusion protein (e.g., the tumor-associated antigen binder) are described; a TAA binder is coupled to a blocking domain via a protease-cleavable linker.

[0370] The protease-cleavable linker is engineered to comprise a sequence recognized by a protease typically present in the tumor microenvironment (TME). The tumor microenvironment is characterized by numerous factors including but not limited to expression of matrix metalloproteinases, cathepsins, proteases, indolomine 2,3-dioxygenase, urokinase-type plasminogen activator (uPA), membrane-type protease 1 (MT-SPl/matriptase), legumain, low extracellular pH (acidosis) and low oxygenation (hypoxia) (Brown & Wilson, 2004; Vaupel & Mayer, 2007; Desnoyers & Vasiljeva, 2013).

[0371] In one embodiment, the protease-cleavable linker comprises a sequence recognized by matrix metalloproteinases (MMPs), which are inactive in non-cancerous cells, thereby preventing normal activity of the polypeptide fusion protein until the linker is cleaved, and the masking domain is therefore removed, by TME -expressed MMPs. Non-limiting examples of MMP cleavage sites are listed in Table 14.

Table 14: Exemplary MMP-cleavable sequences

[0372] The linker comprising the protease -cleavable sequence is covalently attached to a blocking domain that physically blocks or sterically inhibits the biologically active polypeptide from functioning in non-cancerous tissue. In one embodiment, a bulky protein such as human serum albumin (HSA, or a fragment thereof) is used. Such bulky proteins can block access of an active protein to its target or prevent binding of an antigen to its receptor.

[0373] The preparation comprising the biologically active fusion protein, protease-cleavable linker, and blocking domain remains inactive until it reaches the tumor microenvironment, at which point the linker is cleaved by proteases present and the cytokine, chemokine, or binding agent is unmasked and/or becomes active.

[0374] Successful cleavage by TME proteases in vivo may be assessed in several ways. In one embodiment, a xenograft model system is used comprising an inoculation cell line that expresses the protease of interest and as a negative control an inoculation cell line that does not express the protease of interest is used (e.g., a similar or same cell line where the gene for the protease is knocked out). When tumors in the two types of mice (i.e., experimental mice and control mice) have grown to a certain size (e.g., about 300mm ² ), infusion of TFP T cells and subsequent measurement of tumor volume, cytokine production, and survival responses will provide a measurement of TFP efficacy and linker cleavage in protease null and positive TMEs.

[0375] In another embodiment, linker cleavage is tested by the harvest and co-IP of TIES expressing a TFP disclosed herein from a) a TAA+ and protease -expressing xenograft and b) TAA+ and protease null xenograft, followed by western blot analysis or intact mass spectrometry to detect linker cleavage.

[0376] In another embodiment, the blocking domain (e.g., an anti-HSA domain) is fused to an epitope (e.g. FLAG, myc, or HA tag) for immunoprecipitation and linker cleavage is measured by detection of the released blocking domain by western blot, ELISA, or mass spectrometry. In this system, TAA+ tumors with and without the relevant protease are compared. The +/- protease cell line system may be achieved by genetic knockout, knock-in, null vs over-expressing lines, or by infusion of a relevant protease.

Example 6. Protease Inducible TFP T cells using Cathepsin-Cleavable Blocking Domains

[0377] This Example describes using linker peptide sequences to attach the blocking domain that are recognized and subsequently digested by cathepsins. Cathepsins are a group of serine proteases that function in acidic environments. Lysosomal cathepsins require a reducing, slightly acidic environment, such as found in the TME, in order to be optimally active.

[0378] A polypeptide fusion protein with a blocking domain is engineered as described in Example 5. A linker comprising a cathepsin cleavable elastin, collagen or fibronectin is linked to the fusion protein in a fashion that prohibits binding domain recognition and/or biological activity. In one embodiment, cathepsin S cleavable binding domains such as GAVVRGA (SEQ ID NO: ) may be used to link the fusion protein to the masking domain (e.g., HSA or a fragment thereof). The cathepsin cleavable blocking domain will be removed in the tumor microenvironment using appropriate cleavable linker sequences (e.g., cathepsin B or cathepsin S sequences).

[0379] The biologically active fusion protein may additionally comprise a serum half-life extension element, as is evidenced by its prolonged activity or extended PK. In one embodiment, the serum half- life extension element is the HSA blocking domain. In some embodiments, the serum half-life extension element may extend the serum half-life of a T cell comprising a biologically active fusion protein.

Example 7. Protease Inducible CAR T cells

[0380] In this Example, a biologically active fusion protein, e.g., a chimeric antigen receptor CAR) subunit, is engineered to comprise a blocking domain as described above. A protease-cleavable linker is attached to each variable domain on the antibody, and each linker is in turn covalently attached to a masking domain, such as HSA or a fragment thereof (see Figures 1A and IB). The masked chimeric antigen receptor is expressed in an immune cell, such as a Treg or NK cell, which is in turn administered to a patient. Upon the engineered cell reaching the tumor microenvironment, the masking domain is cleaved away and the CAR T cells are capable of immunomodulation.

Example 8. Protease Inducible TFP T cells comprising an HSA binding domain

[0381] In this Example, a chimeric antigen receptor subunit (such as described in Example 7) comprises a tumor-associated antigen (TAA) binding protein. The subunit is expressed such that it incorporates into an immune cell ' s endogenous T cell receptor complex.

[0382] The TFP T cell activity is masked by a cleavable HSA binding domain, such as a single domain antibody (sdAb), Fab fragment, V _H , V _L , or scFv, which is fused to the TAA binder (Figure 2A). The HSA binding domain is covalently attached to the TAA binder via a cleavable domain, such as those described above (e.g., MMP14, MMP9, cathepsins, etc.). An exemplary humanized anti-HSA sdAb that may be fused to a TCR T cell that will recognize and bind HSA is

EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLY ADSV KGRFTISRDNAKTTFYFQMNSFRPEDTAVYYCTIGGSFSRSSQGTFVTVSS (SEQ ID NO:42).

[0383] Upon infusion of engineered immune cells comprising this construct into a patient, circulating HSA in the patient will bind to the HSA -binding domain of the construct, resulting in inhibition of the tumor recognition domain. Masking of the TAA-binding domain by the bound HSA reduces the risk of on-target, off-tumor activity of the engineered immune cells. The albumin -binding domain and the bound albumin serve to both extend the serum half-life of the circulating cells and to block the function of the TAA-binding domain.

[0384] Upon migration into the tumor microenvironment, the cleavable domain between the HSA masking domain and the TAA-binding domain is cleaved, and the HSA/HSA-binder complex is removed from the surface of the immune cell. In turn, the tumor recognition domain of the complex is exposed and able to recognize tumor antigen. Example 9. Protease Inducible TFP T cells comprising bound HSA

[0385] In this Example, inducible TFP T cells are prepared as described in Example 8.

[0386] The TFP T cell activity is masked by a cleavable domain comprising a blocking moiety, e.g., HSA or a fragment thereof. The HSA or fragment thereof is either fused directly to the TAA binder or is fused to another part of the TCR in a way that blocks the TAA binding site (Figure 5). The HSA is covalently attached to the TFP via a cleavable domain, such as those described above.

[0387] Upon infusion of engineered immune cells comprising this construct into a subject, the TFP T cells migrate into the tumor microenvironment, and the linker is cleaved by proteases present in the tumor microenvironment, and the HSA is removed from the surface of the immune cell. In turn, the tumor recognition domain of the complex is exposed and able to recognize tumor antigen.

Example 10. Anti-mesothelin inducible TFP constructs

[0388] The inducible TFP constructs disclosed herein can comprise an anti-mesothelin binding domain. Examples of anti-mesothelin binding domain sequences are listed in Table 15.

Table 15. Examples of anti-mesothelin sequences

Digestion of aAlb-linker-SDl fusion protein constructs

[0389] The single domain antibody fusion proteins (sequences are shown in FIG. 6) were expressed in E. coli and purified by Ni-NTA affinity chromatography. Each protein was subjected to cleavage by incubation with their respective proteases (MMP9, uPA or cathepsin B) for the cleavage sequence engineered between the two sdAbs. 850 pmol each of an aHSA sdAb-linker-cleavage site-linker-SD 1 sdAb-His6 construct fusion protein shown in Figure 6 was digested with 0.27 pmol MMP9, 0.22 pmol uPA or 0.18 pmol cathepsin B, corresponding to 2xlO ⁵ U of each protease, for 16 hours at 23 _° C in the presence or absence of 10% fetal bovine serum (FBS). Next, 5 qg of each purified fusion protein subjected to proteolysis was analyzed by western blot using a 1:500 anti-6His HRP antibody

(ThermoFisher Cat. MA1-21315-HRP) in PBST bufferto confirm cleavage products.

[0390] Results are shown in FIG. 8. Constructs were loaded pairwise as follows: aHSA-sdAb-uPA-SD 1 sdAb digested with uPA +/- FBS; aAlb-N/C-SDl digested with uPA +/- FBS; aHSA sdAb-MMP9vl- SDlsdAb digested with MMP9 +/- FBS; aHSA sdAb-MMP9v2-SDlsdAb digested with MMP9 +/- FBS; and aHSA sdAb-cathepsin B-SD1 sdAb digested with cathepsin B +/- FBS. In this western blot, purified fusion proteins transferred to the PVDF membrane were probed with the anti-6His antibody for detection of both the undigested fusion protein with a C-terminal 6His tag (~29 kDa MW) and the cleavage product - SD1 sdAb-His6 (~ l4kDa). As can be seen in Figure 8, the undigested product is approximately 30 kDa and the expected cleavage product is about 14 kDa.

[0391] uPA and MMP9 cleavage product is visualized on the western blot. Cathepsin B either did not cleave or the cleavage was so inefficient that the cleavage product was at a concentration that is below the limit of detection by this method. The efficiency of protease cleavage is quite low, representing 5% or less of the product detected on the western blot membrane. The uPA protease is unable to cleave in the presence of FBS, while MMP9 seems to not be affected by the presence of FBS.

[0392] MMP9vl cleavage sequence seems to be more efficiently cleaved compared to MMP9v2.

Endnotes

[0393] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.