COMPOSITIONS AND METHODS FOR TCR REPROGRAMMING USING FUSION

PROTEINS

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/277,938, filed November 10, 2021, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Most patients with hematological malignancies or with late-stage solid tumors are incurable with standard therapy. In addition, traditional treatment options often have serious side effects. Numerous attempts have been made to engage a patient’s immune system for rejecting cancerous cells, an approach collectively referred to as cancer immunotherapy. However, several obstacles make it rather difficult to achieve clinical effectiveness. Although hundreds of so-called tumor antigens have been identified, these are often derived from self and thus can direct the cancer immunotherapy against healthy tissue, or are poorly immunogenic. Furthermore, cancer cells use multiple mechanisms to render themselves invisible or hostile to the initiation and propagation of an immune attack by cancer immunotherapies.

[0003] Recent developments using chimeric antigen receptor (CAR) modified autologous T cell therapy, which relies on redirecting genetically engineered T cells to a suitable cell-surface molecule on cancer cells, show promising results in harnessing the power of the immune system to treat cancers. For example, the clinical results from an ongoing trial with B-cell maturation antigen (BCMA) -specific CAR T cells have shown partial remission in some multiple myeloma patients (one such trial may be found via clinicaltrials.gov identifier NCT02215967). An alternative approach is the use of T cell receptor (TCR) alpha and beta chains selected for a tumor-associated peptide antigen for genetically engineering autologous T cells. These TCR chains will form complete TCR complexes and provide the T cells with a TCR for a second defined specificity. Encouraging results were obtained with engineered autologous T cells expressing NY-ESO-1 -specific TCR alpha and beta chains in patients with synovial carcinoma.

[0004] Besides the ability of genetically modified T cells expressing a CAR or a second TCR to recognize and destroy respective target cells in vitro/ex vivo, successful patient therapy with engineered T cells requires the T cells to be capable of strong activation, expansion, persistence over time, effective tumor targeting, and, in case of relapsing disease, to enable a ‘memory’ response. High and manageable clinical efficacy of CAR T cells is currently limited to mesothelin-positive B cell malignancies and to NY-ESO-1 -peptide-expressing synovial sarcoma patients expressing HLA-A2. There is a clear need to improve genetically engineered T cells to more broadly act against various human malignancies and to enhance longevity of genetically engineered T cells to generate durable responses in cancer patients.

SUMMARY

[0005] Described herein are novel fusion proteins of TCR subunits, including CD3 epsilon, CD3 gamma and CD3 delta, and of TCR alpha and TCR beta chains with binding domains specific for cell surface antigens that have the potential to overcome limitations of existing approaches. Described herein are novel fusion proteins that more efficiently kill target cells than CARs, but release comparable or lower levels of pro-inflammatory cytokines. These fusion proteins and methods of their use represent an advantage for TFPs relative to CARs because elevated levels of these cytokines have been associated with dose-limiting toxicities for adoptive CAR-T therapies.

[0006] Described herein are recombinant nucleic acids comprising nucleic acids encoding T cell receptor (TCR) fusion proteins (TFPs) specific for CD70 and mesothelin (MSLN), and T cells comprising such recombinant nucleic acids and TFPs. Described herein are TFPs that efficiently kill target cells that express CD70 or MSLN or both CD70 and MSLN. Fusion proteins and methods of their use as described herein represent an advantage relative to CARs or other engineered TCR therapies in their ability to efficiently target both CD70 and MSLN.

[0007] As such, provided herein are recombinant nucleic acids comprising: a first nucleic acid sequence encoding binding proteins having specificity for more than one target, and antibodies and T cell receptor (TCR) fusion proteins (TFPs) comprising such dual-specificity binding proteins. In addition are provided T cells engineered to express one or more TFPs, and methods of use thereof for the treatment of diseases. The TFPs may have dual specificity on a single molecule, or in a single engineered TCR; alternatively, the dual specificity may come from mixing two engineered T cell populations comprising the TFPs, or transducing a single population of T cells with two different viruses.

[0008] In one aspect, inter alia, provided herein is a composition comprising (I) a first recombinant nucleic acid sequence encoding a first T cell receptor (TCR) fusion protein (TFP) comprising (a) a first TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising an intracellular domain derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain; and (b) a first antibody domain comprising an anti-CD70 binding domain, wherein the first TCR subunit and the anti-CD70 binding domain are operatively linked, wherein the first TFP functionally incorporates into an endogenous TCR complex when expressed in a T cell; and (II) a second recombinant nucleic acid sequence encoding a second TFP comprising (a) a second TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising an intracellular domain derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain; and (b) a second antibody domain comprising an anti-mesothelin (MSLN) binding domain, wherein the second TCR subunit and the anti-MSLN binding domain are operatively linked, wherein the second TFP functionally incorporates into an endogenous TCR complex when expressed in a T cell.

[0009] In another aspect, provided herein is a composition comprising (I) a first recombinant nucleic acid sequence encoding a first T cell receptor (TCR) fusion protein (TFP) comprising (a) a first TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising an intracellular domain derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain; and a first antibody domain comprising an anti-CD70 binding domain and a second antibody domain comprising an anti-MSLN binding domain; wherein the first TCR subunit, the first antibody domain, and the second antibody domain are operatively linked, and wherein the first TFP functionally incorporates into an endogenous TCR complex when expressed in a T cell.

[0010] In another aspect, provided herein is a composition comprising a recombinant nucleic acid molecule encoding: (a) a first T cell receptor (TCR) fusion protein (TFP) comprising a first TCR subunit, a first antibody domain comprising a first antigen binding domain that is an anti-CD70 binding domain; and (b) a second T cell receptor (TCR) fusion protein (TFP) comprising a second TCR subunit, a second antibody domain comprising a second antigen binding domain that is an anti- MSLN binding domain, wherein the first TCR subunit of the first TFP and the first antibody domain are operatively linked and the second TCR subunit of the second TFP and the second antibody domain are operatively linked.

[0011] In another aspect, provided herein is a composition comprising a recombinant nucleic acid molecule encoding: a first T cell receptor (TCR) fusion protein (TFP) comprising a first TCR subunit, a first antibody domain comprising a first antigen binding domain that is an anti-CD70 binding domain and a second antibody domain comprising a second antigen binding domain that is an anti- MSLN binding domain; and wherein the first TCR subunit of the first TFP, the first antibody domain and the second antibody domain are operatively linked.

[0012] In some embodiments, the first TCR subunit or the second TCR subunit comprises at least a portion of a TCR extracellular domain, a transmembrane domain, and a TCR intracellular domain. [0013] In some embodiments, the TCR intracellular domain is an intracellular domain derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain.

[0014] In some embodiments, the transmembrane domain is a TCR transmembrane domain from a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain.

[0015] In some embodiments, the TCR extracellular domain is a full-length extracellular domain from a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain.

[0016] In some embodiments, the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit or the second TCR subunit are derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain.

[0017] In some embodiments, the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit or the second TCR subunit are derived only from a TCR subunit selected from the group consisting of a CD3 gamma chain and a CD3 epsilon chain.

[0018] In some embodiments, the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit or the second TCR subunit are derived only from a CD3 gamma chain.

[0019] In some embodiments, the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit or the second TCR subunit are derived only from a CD3 epsilon chain.

[0020] In some embodiments, the first TCR subunit and the second TCR subunit are from the same TCR subunit, and wherein the same TCR subunit is a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain or a CD3 epsilon chain. [0021] In some embodiments, the same TCR subunit is CD3 epsilon chain.

[0022] In some embodiments, the first TCR subunit and the second TCR subunit are different.

[0023] In some embodiments, the first TCR subunit is from a CD3 epsilon chain, and the second TCR chain is from a CD3 gamma chain.

[0024] In some embodiments, the first TCR subunit is from a CD3 gamma chain, and the second TCR chain is from a CD3 epsilon chain.

[0025] In some embodiments, the first TFP and the second TFP incorporate into a same endogenous TCR complex when expressed in a T cell.

[0026] In some embodiments, the first antibody domain or the second antibody domain is a murine, human or humanized antibody domain.

[0027] In some embodiments, the first TCR subunit or the second 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.

[0028] In some embodiments, the first recombinant nucleic acid sequence and the second recombinant nucleic acid sequence are on the same nucleic acid molecule.

[0029] In some embodiments, the first nucleic acid sequence and the second recombinant nucleic acid sequence are on different nucleic acid molecule.

[0030] In some embodiments, (i) the first antibody domain is connected to the TCR extracellular domain of the first TFP by a first linker sequence, (ii) the second antibody domain is connected to the TCR extracellular domain of the second TFP by a second linker sequence, or (iii) both the first antibody domain is connected to the TCR extracellular domain of the first TFP by a first linker sequence and the second antibody domain is connected to the TCR extracellular domain of the second TFP by a second linker sequence.

[0031] In some embodiments, the first antibody domain is connected to the N-terminus of the TCR extracellular domain of the first TFP by the first linker sequence. [0032] In some embodiments, the second antibody domain is connected to the N-terminus of the TCR extracellular domain of the second TFP by the second linker sequence.

[0033] In some embodiments, the first linker sequence or the second linker sequence comprises the sequence of AAAGGGGSGGGGSGGGGSLE (SEQ ID NO: 387).

[0034] In some embodiments, the first antibody domain and the second antibody domain are linked by a first linker sequence.

[0035] In some embodiments, the first antibody domain or the second antibody domain is linked to the first TCR subunit by a second linker sequence.

[0036] In some embodiments, the first linker sequence comprises the sequence of GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 1020).

[0037] In some embodiments, the second linker sequence comprises (G4S) _n , wherein n=l to 4.

[0038] In some embodiments, the first antibody domain is connected to the N-terminus of the second antibody domain, and wherein the second antibody domain is connected to the N-terminus of the first TCR subunit.

[0039] In some embodiments, the second antibody domain is connected to the N-terminus of the first antibody domain, and wherein the first antibody domain is connected to the N-terminus of the first TCR subunit.

[0040] In some embodiments, the first TCR subunit of the first TFP, the second TCR subunit of the second TFP, or both comprise an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of 4- IBB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.

[0041] In some embodiments, the first antibody domain, the second antibody domain, or both comprise an antibody fragment.

[0042] In some embodiments, the first antibody domain, the second antibody domain, or both comprise a scFv or a VH domain.

[0043] In some embodiments, the second antibody domain comprises at least two anti-MSLN binding domains.

[0044] In some embodiments, each of the at least two anti-MSLN binding domains is a VH domain.

[0045] In some embodiments, a first anti-MSLN binding domain is connected to a second anti- MSLN binding domain by a linker.

[0046] In some embodiments, the linker comprises the sequence of

TLGMDELYKSGIRGGGGSGGGGSGGGGSTPRMVS (SEQ ID NO: 248).

[0047] In some embodiments, the first anti-MSLN binding domain and the second anti-MSLN binding domain comprise the same sequence.

[0048] In some embodiments, the anti-CD70 binding domain comprises (i) a light chain binding domain comprising a light chain (LC) CDR1 of SEQ ID NO:365, LC CDR2 of SEQ ID NO:366, and LC CDR3 of SEQ ID NO:367, and/or (ii) a heavy chain binding domain comprising a heavy chain (HC) CDR1 of SEQ ID NO: 361, HC CDR2 of SEQ ID NO: 362, and HC CDR3 of SEQ ID NO: 363. [0049] In some embodiments, the anti-CD70 binding domain comprises 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 SEQ ID NO:368, or a sequence with 95-99% identity to a light chain variable region amino acid sequence of SEQ ID NO:368.

[0050] In some embodiments, the anti-CD70 binding domain comprises 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 SEQ ID NO:364, or a sequence with 95-99% identity to a heavy chain variable region amino acid sequence of SEQ ID NO:364.

[0051] In some embodiments, the anti-CD70 binding domain comprises a heavy chain comprising a heavy chain (HC) CDR1 of SEQ ID NO: 104, HC CDR2 of SEQ ID NO: 105 and HC CDR3 of SEQ ID NO: 106.

[0052] In some embodiments, the anti-CD70 binding domain comprises 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 SEQ ID NO: 107, or a sequence with 95-99% identity to a heavy chain variable region amino acid sequence of SEQ ID NO: 107.

[0053] In some embodiments, the anti-MSLN binding domain comprises a heavy chain comprising a heavy chain (HC) CDR1 of SEQ ID NO:60, HC CDR2 of SEQ ID NO:61 and HC CDR3 of SEQ ID NO: 62, or a heavy chain comprising a HC CDR1 of SEQ ID NO: 63, HC CDR2 of SEQ ID NO: 64 and HC CDR3 of SEQ ID NO: 65.

[0054] In some embodiments, the anti-MSLN binding domain comprises 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 SEQ ID NO:69 or SEQ ID NO:70, or a sequence with 95-99% identity to a heavy chain variable region amino acid sequence of SEQ ID NO:69 or SEQ ID NO:70.

[0055] In some embodiments, the first TFP and the second TFP include 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, CD 16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0056] In some embodiments, the composition as described herein further comprises a sequence encoding a costimulatory domain.

[0057] In some embodiments, the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA- 1 (CDl la/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.

[0058] In some embodiments, the recombinant nucleic acid sequence or the recombinant nucleic acid molecule further comprises a sequence encoding an intracellular signaling domain.

[0059] In some embodiments, the recombinant nucleic acid sequence or the recombinant nucleic acid molecule further comprises a leader sequence.

[0060] In some embodiments, the recombinant nucleic acid molecule further comprises a protease cleavage site.

[0061] In some embodiments, the first recombinant nucleic acid sequence and the second recombinant nucleic acid sequence are linked by a sequence encoding a protease cleavage site. [0062] 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 first TFP, the second TFP, or both.

[0063] In some embodiments, the recombinant nucleic acid molecule is an mRNA.

[0064] In some embodiments, the first TFP, the second TFP, or both include 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, CD16a, CD16b, 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.

[0065] In some embodiments, the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon.

[0066] 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.

[0067] In some embodiments, the first TFP or the second TFP does not comprise a costimulatory domain or a heterologous stimulatory domain.

[0068] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, an anti-CD70 binding domain operatively linked to the first TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit are derived only from the CD3 epsilon chain, and the second TFP comprises, from the N-terminus to the C- terminus, an anti-MSLN binding domain operatively linked to the second TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the second TCR subunit are derived only from the CD3 gamma chain.

[0069] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO: 364 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 733, and the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 69 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 734.

[0070] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046, and the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1050 operatively linked to the sequence of SEQ ID NO: 1041.

[0071] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, an anti-CD70 binding domain operatively linked to the first TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit are derived only from the CD3 gamma chain, and the second TFP comprises, from the N-terminus to the C- terminus, an anti-MSLN binding domain operatively linked to the second TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the second TCR subunit are derived only from the CD3 epsilon chain.

[0072] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO: 364 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 734, and the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 69 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 733.

[0073] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1040 operatively linked to the sequence of SEQ ID NO: 1041, and the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1085 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1086 operatively linked to the sequence of SEQ ID NO: 1046.

[0074] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, an anti-CD70 binding domain operatively linked to the first TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit are derived only from the CD3 epsilon chain, and the second TFP, from the N-terminus to the C-terminus, comprises an anti-MSLN binding domain operatively linked to the second TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the second TCR subunit are derived only from the CD3 epsilon chain.

[0075] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO: 364 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 733, and the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 69 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 1018.

[0076] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046, and the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1050 operatively linked to the sequence of SEQ ID NO: 1051.

[0077] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, an anti-CD70 binding domain operatively linked to the first TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit are derived only from the CD3 epsilon chain, and the second TFP comprises, from the N-terminus to the C- terminus, a first anti-MSLN binding domain operatively linked to a second anti-MSLN binding domain operatively linked to the second TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the second TCR subunit are derived only from the CD3 gamma chain.

[0078] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO: 364 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 733, and the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 69 operatively linked to the sequence of SEQ ID NO: 248 operatively linked to the sequence of SEQ ID NO: 69 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 734.

[0079] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046, and the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1056 operatively linked to the sequence of SEQ ID NO: 1057 operatively linked to the sequence of SEQ ID NO: 1050 operatively linked to the sequence of SEQ ID NO: 1041.

[0080] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, an anti-CD70 binding domain operatively linked to the first TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit are derived only from the CD3 gamma chain, and the second TFP comprises, from the N-terminus to the C- terminus, a first anti-MSLN binding domain operatively linked to a second anti-MSLN binding domain operatively linked to the second TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the second TCR subunit are derived only from the CD3 epsilon chain.

[0081] In some embodiments, the first TFP is operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of a T2A linker operatively linked to the N-terminus of the second TFP.

[0082] In some embodiments, the T2A linker comprises the sequence of SEQ ID NO: 23.

[0083] In some embodiments, the T2A linker is encoded by the nucleotide sequence of SEQ ID NO: 1048.

[0084] In some embodiments, the sequence of GSG is encoded by the nucleotide sequence of SEQ ID NO: 1047.

[0085] In some embodiments, the composition comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1003-1009, 1012, 1013, and 1016.

[0086] In some embodiments, the composition comprises any one of the sequences selected from SEQ ID NOs: 1003-1009, 1012, 1013, and 1016.

[0087] In some embodiments, the composition comprises a sequence encoded by a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the nucleotide sequences selected from SEQ ID NOs: 1024-1030, 1033, 1034, and 1037.

[0088] In some embodiments, the composition comprises a sequence encoded by any one of the nucleotide sequences selected from SEQ ID NOs: 1024-1030, 1033, 1034, and 1037.

[0089] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, an anti-CD70 binding domain operatively linked to an anti-MSLN binding domain operatively linked to the first TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit are derived only from the CD3 epsilon chain. [0090] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO: 364 operatively linked to the sequence of SEQ ID NO: 1020 operatively linked to the sequence of SEQ ID NO: 69 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 733.

[0091] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1084 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046.

[0092] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, an anti-MSLN binding domain operatively linked to an anti-CD70 binding domain operatively linked to the first TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit are derived only from the CD3 epsilon chain.

[0093] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 69 operatively linked to the sequence of SEQ ID NO: 1020 operatively linked to the sequence of SEQ ID NO: 368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO: 364 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 733.

[0094] In some embodiments, the TFP is encoded by a nucleotide sequence comprising, from 5 ’-end to 3 ’-end, the sequence of SEQ ID NO: 1055 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1084 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046.

[0095] In some embodiments, the first TFP comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 1014 or SEQ ID NO: 1015.

[0096] In some embodiments, the first TFP comprises the sequence of SEQ ID NO: 1014 or SEQ ID NO: 1015.

[0097] In some embodiments, the first TFP is encoded by a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 1035 or SEQ ID NO: 1036.

[0098] In some embodiments, the first TFP is encoded by the sequence of SEQ ID NO: 1035 or SEQ ID NO: 1036. [0099] In another aspect, provided herein is a composition comprising a polypeptide molecule encoded by the recombinant nucleic acid molecule of the composition as described herein.

[0100] In some embodiments, the polypeptide comprises a first polypeptide encoded by a first nucleic acid molecule and a second polypeptide encoded by a second nucleic acid molecule.

[0101] In another aspect, provided herein is a composition comprising a recombinant TFP molecule encoded by the recombinant nucleic acid molecule of the composition as described herein.

[0102] In another aspect, provided herein is a composition comprising a vector comprising a nucleic acid sequence encoding the polypeptide or recombinant TFP molecule as described herein.

[0103] In some embodiments, the vector comprises a) a first vector comprising a first nucleic acid sequence encoding the first TFP; and b) a second vector comprising a second nucleic acid sequence encoding the second TFP.

[0104] In some embodiments, the vector is selected from the group consisting of a DNA, an RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector.

[0105] In some embodiments, the composition as described herein further comprises a promoter.

[0106] In some embodiments, the vector is an in vitro transcribed vector.

[0107] In some embodiments, the nucleic acid molecule in the vector further encodes a poly(A) tail.

[0108] In some embodiments, the nucleic acid molecule in the vector further encodes a 3’UTR.

[0109] In some embodiments, the nucleic acid molecule in the vector further encodes a protease cleavage site.

[0110] In another aspect, provided herein is a composition comprising a cell comprising the composition as described herein.

[oni] In some embodiments, the cell is a human T cell.

[0112] In some embodiments, the T cell is a CD8+ or CD4+ T cell.

[0113] In some embodiments, the composition as describe herein 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.

[0114] In some embodiments, the inhibitory molecule comprises a first polypeptide that comprises at least a portion of PD 1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.

[0115] In another aspect, provided herein is a vector comprising the recombinant nucleic acid sequence as described herein.

[0116] In another aspect, provided herein is a vector comprising the first recombinant nucleic acid sequence as described herein.

[0117] In another aspect, provided herein is a vector comprising the second recombinant nucleic acid sequence as described herein.

[0118] In another aspect, provided herein is a cell comprising the composition as described herein or the vector as described herein.

[0119] In some embodiments, the cell is a human T cell.

[0120] In some embodiments, the human T cell is a CD8+ or CD4+ T cell.

[0121] In some embodiments, the cell is an alpha beta T cell.

[0122] In some embodiments, the cell is not a gamma delta (y5) T cell.

[0123] In some embodiments, the cell is not a V 51+ V 52- y5 T cell, not a V 5 1- V 52+ y5 T cell or not a V 51- V 52- yS T cell.

[0124] In some embodiments, the cell as described herein 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.

[0125] In some embodiments, the inhibitory molecule comprises a first polypeptide that comprises at least a portion of PD 1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.

[0126] In some embodiments, the cell as described herein further comprises a nucleic acid encoding an IL- 15 polypeptide or a fragment thereof. In some embodiments, the cell as described herein further comprises a nucleic acid encoding an IL-15Ra polypeptide or fragment thereof. In some embodiments, the cell as described herein further comprises a nucleic acid encoding a fusion protein comprising an IL- 15 polypeptide or a fragment thereof linked to an IL-15Ra polypeptide or a fragment thereof. In some embodiments, the IL- 15 polypeptide or the IL- 15 fusion protein is expressed on cell surface (i.e., is membrane bound) when expressed in a cell, e.g., a T cell. In some embodiments, the IL- 15 polypeptide or the IL- 15 fusion protein is secreted when expressed in a cell, e.g., a T cell.

[0127] In another aspect, provided herein is a protein complex comprising: (i) a first TFP molecule comprising an anti-CD70 binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; (ii) a second TFP molecule comprising an anti-MSLN binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and (iii) at least one endogenous TCR subunit or endogenous TCR complex.

[0128] In another aspect, provided herein is a protein complex comprising: (i) a TFP molecule comprising an anti-CD70 binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and (ii) at least one endogenous TCR subunit or endogenous TCR complex. [0129] In another aspect, provided herein is a protein complex comprising: (i) a TFP molecule comprising an anti-MSLN binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and (ii) at least one endogenous TCR subunit or endogenous TCR complex.

[0130] 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. [0131] In some embodiments, the anti-CD70 binding domain, the anti-MSLN binding domain, or both are connected to the TCR extracellular domain by a linker sequence.

[0132] In some embodiments, the linker region comprises (G4S) _n , wherein n=l to 4.

[0133] In another aspect, provided herein is a human CD8+ or CD4+ T cell comprising at least two different TFP proteins per the protein complex as described herein.

[0134] In another aspect, provided herein is a human CD8+ or CD4+ T cell comprising at least two different TFP molecules encoded by the isolated nucleic acid molecule as described herein.

[0135] In another aspect, provided herein is a population of human CD8+ or CD4+ T cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules, the TFP molecules comprising an anti-CD70 binding domain or an anti-MSLN binding domain, or both an anti-CD70 and an anti-MSLN 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+ T cell.

[0136] In another aspect, provided herein is a population of human CD8+ or CD4+ T cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules encoded by the recombinant nucleic acid molecule as described herein.

[0137] In another aspect, provided herein is a pharmaceutical composition comprising an effective amount of the composition as described herein, the vector as described herein, the cell as described herein, or the protein complex as described herein, and a pharmaceutically acceptable excipient.

[0138] In another aspect, provided herein is a pharmaceutical composition comprising an effective amount of the cell as described herein, the population as described herein, and a pharmaceutically acceptable excipient.

[0139] In another aspect, provided herein is a method of treating a mammal having a disease associated with expression of MSLN or CD70 comprising administering to the mammal an effective amount of the composition as described herein.

[0140] In some embodiments, the disease associated with CD70 or MSLN expression is selected from the group consisting of mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer), ovarian cancer, prostate cancer, colon cancer, colorectal cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, renal cancer, thyroid cancer, bladder cancer, ureter cancer, kidney cancer, endometrial cancer, esophageal cancer, gastric cancer, skin cancer, thymic carcinoma and cholangiocarcinoma.

[0141] In some embodiments, the disease associated with CD70 or MSLN expression is malignant pleural mesothelioma (MPM), ovarian cancer, renal cell carcinoma, or acute myeloid leukemia (AML).

[0142] In some embodiments, the disease is a hematologic cancer selected from the group consisting of B-cell acute lymphoid leukemia (B-ALL), T cell acute lymphoid leukemia (T-ALL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell-follicular lymphoma, large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin’s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and preleukemia.

[0143] In some embodiments, the cells expressing a first TFP molecule and a second TFP molecule are administered in combination with an agent that increases the efficacy of a cell expressing the first TFP molecule and the second TFP molecule.

[0144] In some embodiments, less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing: (a) an anti-MSLN chimeric antigen receptor (CAR); (b) an anti-CD70 CAR; (c) an anti-MSLN CAR and an anti-CD70 CAR; or (d) a combination thereof.

[0145] In some embodiments, the cells expressing the first TFP molecule and a second TFP molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing the first TFP molecule and the second TFP molecule.

[0146] In some embodiments, the cells expressing the first TFP molecule and a second TFP molecule are administered in combination with an agent that treats the disease associated with MSLN or CD70.

INCORPORATION BY REFERENCE

[0147] 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

[0148] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure”, “Fig.”, and “FIGURE” herein) of which:

[0149] Figure 1 shows schematic illustrations of exemplary embodiments of the dual TRuC and tandem binder constructs described herein.

[0150] Figure 2 shows flow cytometry data collected from T cells transduced with CD70 and/or MSLN targeting mono or dual TRuCs.

[0151] Figure 3 shows percent cytotoxicity of target 786-0, MSTO-MSLN, or 786-O:MSTO-MSLN cells after co-culture with T cells transduced with CD70 and/or MSLN targeting mono or dual TRuCs.

[0152] Figure 4 shows flow cytometry data collected from T cells transduced with CD70 and/or MSLN targeting mono or dual TRuCs. [0153] Figure 5 shows percent cytotoxicity of target MSTO-MSLN, 786-0, OVCAR8 and U2OS cells after co-culture with T cells transduced with CD70 and/or MSLN targeting mono or dual TRuCs.

[0154] Figure 6 shows flow cytometry data collected from CD4+ T cells transduced with CD70 and/or MSLN targeting mono or dual TRuCs and associated memory phenotype quantification.

[0155] Figure 7 shows flow cytometry data collected from CD8+ T cells transduced with CD70 and/or MSLN targeting mono or dual TRuCs and associated memory phenotype quantification.

[0156] Figure 8A and Figure 8B show the tumor volume (mm ³ ) over time in MSTO-MSLN (Figure 8A) or 786-0 (Figure 8B) tumor bearing mice after administration of T cells transduced with CD70 targeting mono TRuCs (ClOe), MSLN targeting mono TRuCs (MHlg, TC-210), or CD10/MSLN targeting dual TRuCs (C10e-T2A-MHlg), or after administration of non-transduced T cells (NT) or vehicle control.

[0157] Figure 9A shows the fold expansion over time of non-transduced (NT) T cells or T cells transduced with CD70 targeting mono TRuCs (ClOe), MSLN targeting mono TRuCs (MHlg, TC- 210), or CD70/MSLN targeting dual TRuCs (C10e-T2A-MHlg, C10e-T2A-MHle). Figure 9B shows the tumor volume (mm ³ ) in MSTO-MSLN tumor bearing mice after administration of T cells transduced with CD70 targeting mono TRuCs (ClOe), MSLN targeting mono TRuCs (MHlg, TC- 210), or CD70/MSLN targeting dual TRuCs (C10e-T2A-MHlg, C10e-T2A-MHle); or after administration of NT or vehicle control.

[0158] Figure 10 shows the fold expansion overtime of non-transduced (NT) T cells or T cells transduced with CD70/MLSN dual TRuCs C10e-T2A-MHlg, C10e-T2A-MHlg Opti 1, C10e-T2A- MHlg Opti 2, ClOe-MHl tandem, MH1-C10 tandem, or C10g-T2A-MHle.

[0159] Figure 11 shows flow cytometry data collected from T cells from a representative donor, transduced with the indicated CD70/MSLN dual TRuCs. For each flow plot, the X axis indicates expression of MH1 (VHH+) and the Y axis indicates expression of CIO. The MFI for MH1 in each group is provided below each flow plot. MFI for C10a-T2A-MHly Optil is not applicable as surface expression as not detected.

[0160] Figure 12 shows flow cytometry histogram plots from T cells from a representative donor, transduced with the indicated CD70/MSLN dual TRuCs. The table in the figure provides the MFI for CIO expression in each group.

[0161] Figure 13 shows percent cytotoxicity of target 786-0, MSTO-MSLN, OVCAR8 and U2OS cells after co-culture at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs Opti2 (i.e., C10e-T2A-MHlg Opti2), C10-MH1 tandem, MH1-C10 tandem, ClOg- T2A-MHle, or C10e-T2A-MHlg.

[0162] Figure 14A and Figure 14B show the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of MSTO-MSLN tumor cells at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs Opti2 (i.e., C10e-T2A-MHlg Opti2), C10-MH1 tandem, MH1-C10 tandem, C10g-T2A-MHle, or C10e-T2A-MHlg, for two representative donors.

[0163] Figure 15A and Figure 15B show the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of 786-0 tumor cells at a 3: l, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs Opti2 (i.e., C10e-T2A-MHlg Opti2), C10-MH1 tandem, MH1-C10 tandem, C10g-T2A-MHle, or C10e-T2A-MHlg, for two representative donors.

[0164] Figure 16A and Figure 16B show the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of OVCAR8 tumor cells at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs Opti2 (i.e., C10e-T2A-MHlg Opti2), C10-MH1 tandem, MH1-C10 tandem, C10g-T2A-MHle, or C10e-T2A-MHlg, for two representative donors. [0165] Figure 17A and Figure 17B show the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of U2OS tumor cells at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs Opti2 (i.e., C10e-T2A-MHlg Opti2), C10-MH1 tandem, MH1-C10 tandem, C10g-T2A-MHle, or C10e-T2A-MHlg, for two representative donors.

[0166] Figure 18 shows the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of MSTO-MSLN tumor cells at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells from athird donor transduced with CD70/MSLN dual targeting TRuCs Opti2 (i.e., C10e-T2A-MHlg Opti2), C10-MH1 tandem, MH1-C10 tandem, or C10e-T2A-MHlg, or mono targeting TRuCs ClOe or MHlg.

[0167] Figure 19 provides flow cytometry data showing VHH (anti-MSLN binding domain) expression and Fab (anti-CD70 binding domain) expression on the surface of T cells transduced with C10e-T2A-MHlg, C10e-P2A-MHlg, C10e-fP2A-MHlg, MHl-MHlg, or C10e-T2A-(MHl_MHl)g. [0168] Figure 20 shows percent cytotoxicity of target 786-0, MSTO-MSLN, OVCAR8 and U2OS cells after co-culture at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A-MHlg, C10e-P2A-MHlg, or C10e-T2A-(MHl_MHl)g, or with mono targeting TRuCs MHl-MHlg, ClOe, or MHlg.

[0169] Figure 21A shows the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of 786-0 tumor cells at a 3: l, 1 : 1, or 1 :3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A-MHlg, C10e-P2A-MHlg, or C10e-T2A-

(MHl MHl)g, or with mono targeting TRuCs MHl-MHlg, ClOe, or MHlg. Figure 21B shows the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of MSTO-MSLN tumor cells at a 3: l, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A-MHlg, C10e-P2A-MHlg, or C10e-T2A-(MHl_MHl)g, or with mono targeting TRuCs MHl-MHlg, ClOe, or MHlg. Figure 21C shows the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of OVCAR8 tumor cells at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A-MHlg, C10e-P2A-MHlg, or C10e-T2A-(MHl_MHl)g, or with mono targeting TRuCs MHl-MHlg, ClOe, or MHlg. Figure 21D shows the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of U2OS tumor cells at a 3: l, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A-MHlg, C10e-P2A-MHlg, or C10e-T2A-(MHl_MHl)g, or with mono targeting TRuCs MHl-MHlg, ClOe, or MHlg.

[0170] Figure 22 shows schematic illustrations of exemplary dual TRuCs described herein exhibiting favorable in vitro and in vivo properties.

[0171] Figure 23A and Figure 23B show the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of MSTO-MSLN (Figure 23A) or 786-0 (Figure 23B) tumor cells at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A- MHlg, Opti2 (i.e., C10e-T2A-MHlg Opti2), MH1-C10 tandem, or C10g-T2A-MHle.

[0172] Figure 24 shows flow cytometry data collected from CD4+ T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A-MHlg, C10e-T2A-MHlg Opti2, MH1-C10 tandem, or C10g-T2A-MHle. The flow plots show CD45RA (y-axis) and CCR7 (x-axis) staining, and the bar graph provides the memory phenotype quantification for CD4+ cells.

[0173] Figure 25 shows flow cytometry data collected from CD8+ T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A-MHlg, C10e-T2A-MHlg Opti2, MH1-C10 tandem, or C10g-T2A-MHle. The flow plots show CD45RA (y-axis) and CCR7 (x-axis) staining, and the bar graph provides the memory phenotype quantification for CD8+ cells.

[0174] Figure 26 shows the tumor volume (mm ³ ) over time in 786-0 (left panel) or MSTO-MSLN (right panel) tumor bearing mice after administration on Day 0 of vehicle control, non-transduced (NT) T cells, or T cells transduced with CD10/MSLN targeting dual TRuCs C10e-T2A-MHlg, ClOe- T2A-MHlg Opti2, MH1-C10 tandem, or C10g-T2A-MHle. For the mice with 786-0 tumors, CD70 mono targeting TRuCs CIO were administered as the mono TRuC control, and for the mice with MSTO-MSLN tumors, MSLN mono targeting TRuCs TC-210 were administered as the mono TRuC control.

DETAILED DESCRIPTION

[0175] Provided herein are compositions of matter and methods of use for the treatment of a disease such as cancer, using dual specificity T cell receptor fusion proteins having activity against both CD70 and mesothelin (MSLN).

[0176] The present disclosure provides, in an aspect, a composition comprising a first recombinant nucleic acid sequence encoding a first T cell receptor (TCR) fusion protein (TFP) comprising (a) a first TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising an intracellular domain derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain; and (b) a first antibody domain comprising an anti-CD70 binding domain, wherein the first TCR subunit and the anti-CD70 binding domain are operatively linked, wherein the first TFP functionally incorporates into an endogenous TCR complex when expressed in a T cell; and a second recombinant nucleic acid sequence encoding a second TFP comprising (a) a second TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising an intracellular domain derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain; and (b) a second antibody domain comprising an anti-mesothelin (MSLN) binding domain, wherein the second TCR subunit and the anti-MSLN binding domain are operatively linked, wherein the second TFP functionally incorporates into an endogenous TCR complex when expressed in a T cell.

[0177] In another aspect, provided herein is a composition comprising a first recombinant nucleic acid sequence encoding a first T cell receptor (TCR) fusion protein (TFP) comprising a first TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising an intracellular domain derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain; and a first antibody domain comprising an anti-CD70 binding domain and a second antibody domain comprising an anti-MSLN binding domain; wherein the first TCR subunit, the first antibody domain, and the second antibody domain are operatively linked, and wherein the first TFP functionally incorporates into an endogenous TCR complex when expressed in a T cell.

[0178] In another aspect, provided herein is a composition comprising a recombinant nucleic acid molecule encoding: a first T cell receptor (TCR) fusion protein (TFP) comprising a first TCR subunit, a first antibody domain comprising a first antigen binding domain that is an anti-CD70 binding domain; and a second T cell receptor (TCR) fusion protein (TFP) comprising a second TCR subunit, a second antibody domain comprising a second antigen binding domain that is an anti-MSLN binding domain, wherein the first TCR subunit of the first TFP and the first antibody domain are operatively linked and the second TCR subunit of the second TFP and the second antibody domain are operatively linked.

[0179] In another aspect, provided herein is a composition comprising a recombinant nucleic acid molecule encoding: a first T cell receptor (TCR) fusion protein (TFP) comprising a first TCR subunit, a first antibody domain comprising a first antigen binding domain that is an anti-CD70 binding domain and a second antibody domain comprising a second antigen binding domain that is an anti- MSLN binding domain; and wherein the first TCR subunit of the first TFP, the first antibody domain and the second antibody domain are operatively linked.

[0180] In another aspect, provided herein is a protein complex comprising: a first TFP molecule comprising an anti-CD70 binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; a second TFP molecule comprising an anti-MSLN binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and at least one endogenous TCR subunit or endogenous TCR complex.

[0181] In another aspect, provided herein is a protein complex comprising: a TFP molecule comprising an anti-CD70 binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and at least one endogenous TCR subunit or endogenous TCR complex. [0182] In another aspect, provided herein is a protein complex comprising: a TFP molecule comprising an anti-MSLN binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and at least one endogenous TCR subunit or endogenous TCR complex. [0183] The present disclosure further provides a vector comprising the recombinant nucleic acid, a cell comprising the recombinant nucleic acid or the vector described herein, or a pharmaceutical composition comprising the cell (e.g., modified cell).

[0184] In some embodiments, provided herein are compositions of matter and methods of use for the treatment of a disease such as cancer, using dual specificity T cell receptor (TCR) fusion proteins or dual specificity T cell populations.

Definitions

[0185] 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 present disclosure pertains.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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 disclosure 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.

[0191] As used herein, an “effective amount” or 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)).

[0192] The term “GMCSFRa,” also known as CSF2RA, CD116, Cluster of Differentiation 116, CDwl l6, CSF2R, CSF2RAX, CSF2RAY, CSF2RX, CSF2RY, GM-CSF-R-alpha, GMCSFR, GMR, SMDP4, colony stimulating factor 2 receptor alpha subunit, alphaGMR, colony stimulating factor 2 receptor subunit alpha, GMR-alpha, GMCSFR-alpha, granulocyte -macrophage colony-stimulating factor receptor, as used herein, refers to a receptor for granulocyte-macrophage colony-stimulating factor, which stimulates the production of white blood cells. In some embodiments, GM-CSF and its receptor play a role in earlier stages of development. In some embodiments, GMCSFRa is associated with Surfactant metabolism dysfunction type 4. GMCSFRa, as used herein, includes any of the recombinant or naturally-occurring forms of GMCSFRa or variants or homologs thereof that have or maintain GMCSFRa activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring GMCSFRa. In some embodiments, GMCSFRa is substantially identical to the protein identified by the UniProt reference number P 15509 or a variant or homolog having substantial identity thereto.

[0193] The term “CD28,” also known as Cluster of Differentiation 28, CD28, Tp44, and CD28 molecule, as used herein, refers to a protein expressed on T cells that provides co-stimulatory signals required for T cell activation and survival. In some embodiments, CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins. “CD28,” as used herein, includes any of the recombinant or naturally-occurring forms of CD28 or variants or homologs thereof that have or maintain CD28 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD28. In some embodiments, CD28 is substantially identical to the protein identified by the UniProt reference number Pl 0747 or a variant or homolog having substantial identity thereto.

[0194] The term “2A” “2A self-cleaving peptide,” or “2A peptide,” as used herein, refers to a class of peptides, which can induce ribosomal skipping during translation of a protein in a cell. These peptides share a core sequence motif of DxExNPGP, and are found in a wide range of viral families. Exemplary members of 2A include, but are not limited to, P2A, E2A, F2A, and T2A. “T2A” refers to the 2A derived from thosea asigna virus, and the sequence is EGRGSLLTCGDVEENPGP (SEQ ID NO:23). “P2A” refers to the 2A derived from porcine teschovirus-1 2A, and the sequence is ATNFSLLKQAGDVEENPGP (SEQ ID NO: 1652). “E2A” refers to the 2A derived from quine rhinitis A virus, and the sequence is QCTNYALLKLAGDVESNPGP (SEQ ID NO: 1653). F2A is derived from foot-and-mouth disease virus 18, and the sequence is VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 1654). In some embodiments, adding the 1 linker “GSG” (Gly-Ser-Gly) on the N-terminal of a 2A peptide helps with efficiency.

[0195] The term “furin cleavage site,” as used herein, refers to a cleavage site recognized by protease enzyme furin, also known as FUR, PACE, PCSK3, SPC1, and paired basic amino acid cleaving enzyme. In some embodiments, furin is a subtilisin-like proprotein convertase family. In some embodiments, furin cleaves proteins just downstream of a basic amino acid target sequence (canonically, Arg-X-(ArgZLys) -Arg').

[0196] As used herein, the terms “cleave” or “cleavage” refer to the hydrolysis of phosphodiester bonds within the backbone of a recognition sequence within a target sequence that results in a doublestranded break within the target sequence, referred to herein as a “cleavage site”.

[0197] 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.

[0198] 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 VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments.

[0199] 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.

[0200] “Heavy chain variable region” or “VH” (or, in the case of single domain antibodies, e.g., nanobodies, “VHH”) 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. [0201] Unless specified, as used herein an scFv may have the VL 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 VL- linker-VH or may comprise Vn-linker-VL. [0202] 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.

[0203] 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 ( ■'/. ") light chains refer to the two major antibody light chain isotypes.

[0204] 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.

[0205] 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.

[0206] 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 disclosure 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.

[0207] 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 present disclosure in prevention of the occurrence of tumor in the first place.

[0208] The term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced into the individual. [0209] 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.

[0210] The term “xenogeneic” refers to a graft derived from an animal of a different species.

[0211] The term “cancer” refers 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, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, esophageal cancer, gastric cancer, unresectable ovarian cancer with relapsed or refractory disease, and the like.

[0212] 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 present disclosure 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 present disclosure 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.

[0213] The term “stimulation,” as used herein, 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.

[0214] The term “stimulatory molecule” or “stimulatory domain,” as used herein, 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 present disclosure 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.

[0215] The term “antigen presenting cell” or “APC,” as used herein, 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.

[0216] The portion of the TFP composition of the present disclosure 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 a single chain antibody (scFv) derived from a murine, humanized or human antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426) or heavy chain antibodies HCAb. In one aspect, the antigen binding domain of a TFP composition of the present disclosure comprises an antibody fragment. In a further aspect, the TFP comprises an antibody fragment that comprises a scFv or a sdAb.

[0217] “Major histocompatibility complex (MHC) molecules,” as used herein, are typically bound by TCRs as part of peptide: MHC complex. The MHC molecule may be an MHC class I or II molecule. The complex may be on the surface of an antigen presenting cell, such as a dendritic cell or a B cell, or any other cell, including cancer cells, or it may be immobilized by, for example, coating on to a bead or plate.

[0218] The “human leukocyte antigen system (HLA),” as used herein, refers to the gene complex which encodes major histocompatibility complex (MHC) in humans and includes HLA class I antigens (A, B & C) and HLA class II antigens (DP, DQ, & DR). HLA alleles A, B and C present peptides derived mainly from intracellular proteins, e.g., proteins expressed within the cell.

[0219] During T cell development in vivo, T cells undergo a positive selection step to ensure recognition of self MHCs followed by a negative step to remove T cells that bind too strongly to MHC which present self-antigens. As a consequence, certain T cells and the TCRs they express will only recognize peptides presented by certain types of MHC molecules - i.e., those encoded by particular HLA alleles. This is known as HLA restriction.

[0220] In some embodiments, one HLA allele of interest is HLA-A*0201, which is expressed in the vast majority (>50%) of the Caucasian population. Accordingly, TCRs which bind WT1 peptides presented by MHC encoded by HLA-A*0201 (i.e. are HLA-A*0201 restricted) are advantageous since an immunotherapy making use of such TCRs will be suitable for treating a large proportion of the Caucasian population.

[0221] In some embodiment, other HLA- A alleles of interest are HLA-A*0101, HLA-A*2402, and HLA-A*0301.

[0222] In some embodiments, widely expressed HLA-B alleles of interest are HLA-B*3501, HLA- B*0702 and HLA-B*3502.

[0223] The term “intracellular signaling domain,” as 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, but are not limited to, cytolytic activity and T helper cell activity, including the secretion of cytokines. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include, but are not limited to, those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In some embodiments, the intracellular signaling domain comprises a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include, but are not limited to, those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.

[0224] In some embodiment, a primary intracellular signaling domain comprises 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.

[0225] The term “costimulatory molecule,” as used herein, 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. In some embodiments, costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Exemplary costimulatory molecules include, but are not limited to, an MHC class 1 molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD2, CD27, CD28, CD5, ICAM-1, LFA-1 (CDIIa/CDI8 4-1BB (CD137), IL-15Ra, IL12R, IL18R, IL21R, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, and FcyRIII. In some embodiments, a costimulatory intracellular signaling domain is the intracellular portion of a costimulatory molecule. In some embodiments, a costimulatory molecule is 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-1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, IL-15Ra, IL12R, IL18R, IL21R, CD27, CD5, ICAM-1, CD7, CD226, FcyRI, FcyRII, FcyRIII, and the like. In some embodiments, the intracellular signaling domain comprises the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.

[0226] The term “4-1BB,” as used herein, refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. 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,” as used herein, refers to amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g. , mouse, rodent, monkey, ape and the like. . “4-1BB,” also known as TNFRSF9, 4-1BB, CD137, Cluster of Differentiation 137, CDwl37, ILA, tumor necrosis factor receptor superfamily member 9, and TNF receptor superfamily member 9, as used herein, includes any of the recombinant or naturally-occurring forms of 4- IBB or variants or homologs thereof that have or maintain 4-1BB activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring 4-1BB. In some embodiments, 4- IBB is substantially identical to the protein identified by the UniProt reference number Q07011 or a variant or homolog having substantial identity thereto.

[0227] 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.

[0228] 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 an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain one or more introns.

[0229] The term “CD3” or “Cluster of Differentiation 3,” as used herein, refers to a protein complex and T cell co-receptor that is involved in activating both the cytotoxic T cell and T helper cells. In some embodiments, it is composed of four distinct chains. For example, in some embodiments, the complex contains a CD3y chain, a CD35 chain, and two CD3a chains in mammals. [0230] ‘ CD3s.' “CD3a chain,” or “T-cell surface glycoprotein CD3 epsilon chain,” as used herein, includes any of the recombinant or naturally-occurring forms of CD3s or variants or homologs thereof that have or maintain CD3a activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD3s. In some embodiments, CD3s is substantially identical to the protein identified by the UniProt reference number P07766 or a variant or homolog having substantial identity thereto. In some embodiments, “e” is used interchangeably with “a” for example to refer to the CD3 epsilon, e.g. the CD3 epsilon subunit to which a binding domain provided herein is linked. For example, “MHle” and "MH Is" may be used interchangeably to refer to an MH1 (anti-MSLN) binding domain linked to a CD3a chain. For example, “ClOe” and “CIOs” may be used interchangeably to refer to a CIO (anti-CD70) binding domain linked to a CD3a chain.

[0231] ‘ ‘CD35,” “CD35 chain,” or “T-cell surface glycoprotein CD3 delta chain,” as used herein, includes any of the recombinant or naturally-occurring forms of CD35 or variants or homologs thereof that have or maintain CD35 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD35. In some embodiments, CD35 is substantially identical to the protein identified by the UniProt reference number P04234 or a variant or homolog having substantial identity thereto.

[0232] “CD3y,” “CD3y chain,” or “T-cell surface glycoprotein CD3 gamma chain,” as used herein, includes any of the recombinant or naturally-occurring forms of CD3y or variants or homologs thereof that have or maintain CD3y activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD3y. In some embodiments, CD3y is substantially identical to the protein identified by the UniProt reference number P09693 or a variant or homolog having substantial identity thereto. In some embodiments, “g” is used interchangeably with “y” for example to refer to the CD3 gamma subunit, e.g., the CD3 gamma subunit to which a binding domain provided herein is linked. For example, “MHlg” and “MHly” may be used interchangeably to refer to an MH1 (anti-MSUN) binding domain linked to a CD3y chain. For example, “ClOg” and “ClOy” may be used interchangeably to refer to a CIO (anti-CD70) binding domain linked to a CD3y chain.

[0233] The term “mesothelin (MSLN),” also known as MPF and SMRP, refers to a tumor differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. In some embodiments, mesothelin is over-expressed in several human tumors, including mesothelioma and ovarian and pancreatic adenocarcinoma. MSLN, as used herein, includes any of the recombinant or naturally-occurring forms of MSLN or variants or homologs thereof that have or maintain MSLN activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring MSLN. In some embodiments, MSLN is substantially identical to the protein identified by the UniProt reference number QI 3421 or a variant or homolog having substantial identity thereto.

[0234] “Programmed cell death protein 1,” also known as PD-1, CD279 (cluster of differentiation 279), PDCD1, PD1, SLEB2, hPD-1, hSLEl, and Programmed cell death 1, refers to a protein on the surface of cells that has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells. PD-1 is an immune checkpoint and guards against autoimmunity, e.g., through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T- cells in lymph nodes. Second, it reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells). PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, PD-L1 and PD-L2. PD-1, as used herein, includes any of the recombinant or naturally-occurring forms of PD-1 or variants or homologs thereof that have or maintain PD-1 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PD-1. In some embodiments, PD-1 is substantially identical to the protein identified by the UniProt reference number Q15116 or a variant or homolog having substantial identity thereto.

[0235] ‘ ‘CD70,” also known as CD27LG and TNFSF7, as referred herein, refers to a cytokine that is the ligand for CD27. The CD70-CD27 pathway plays an important role in the generation and maintenance of T cell immunity, in particular, during antiviral responses. Upon CD27 binding, CD70 induces the proliferation of co-stimulated T-cells and enhances the generation of cytolytic T-cells. CD70, as referred herein, includes any of the recombinant or naturally-occurring forms of CD70 or variants or homologs thereof that have or maintain CD70 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD70. In some embodiments, CD70 is substantially identical to the protein identified by the UniProt reference number P32970 or a variant or homolog having substantial identity thereto.

[0236] The term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0237] The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0238] The term “expression” refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

[0239] The term “transfer vector,” as used herein, 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.

[0240] The term “expression vector,” as used herein, 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.

[0241] The term “lentivirus,” as used herein, 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 lentiviruses. The term “lentiviral vector,” as used herein, 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.

[0242] The term “homologous” or “identity,” as used herein, 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.

[0243] “Humanized” forms of non-human (e.g., murine) antibodies, as described herein, 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.

[0244] “Human” or “fully human” immunoglobulin, as used herein, 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.

[0245] The term “isolated,” as used herein, 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.

[0246] In the context of the present disclosure, 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.

[0247] The term “operably linked” or “transcriptional control,” as used herein, 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.

[0248] The terms “nucleotide,” “nucleic acid” and “polynucleotide,” as used herein, are used interchangeably, and refer 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 al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0249] 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.

[0250] The terms “peptide,” “polypeptide,” and “protein,” as used herein, 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.

[0251] The term “promoter,” as used herein, 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.

[0252] The term “promoter/regulatory sequence,” as used herein, 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.

[0253] The term “constitutive” promoter, as used herein, 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.

[0254] The term “inducible” promoter, as used herein, 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.

[0255] The term “tissue-specific” promoter, as used herein, 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.

[0256] The terms “linker” and “flexible polypeptide linker” 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 n=10. In one embodiment, the flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3. In another embodiment, the linkers include multiple repeats of (Gly2Ser), (GlySer) or (Gly ^Scr). Also included within the scope of the present disclosure are linkers described in WO2012/138475 (incorporated herein by reference). In some instances, the linker sequence comprises (G4S) _n , wherein n=2 to 5. In some instances, the linker sequence comprises (G ₄ S) n, wherein n=l to 3. In some instances, the linker sequence comprises (G4S) _n , wherein n=l to 4. In some instances, the linker sequence comprises (G4S) _n , wherein n=2 to 4.

[0257] 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.

[0258] As used herein, “z« 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.

[0259] As used herein, a “poly(A)” refers to 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.

[0260] 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 (SEQ ID NO: 1655) near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3’ end at the cleavage site.

[0261] 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.

[0262] The term “signal transduction pathway,” as used herein, 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.

[0263] The term, a “substantially purified” cell, as used herein, 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.

[0264] The term “therapeutic” as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.

[0265] The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state.

[0266] In the context of the present disclosure, “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 disclosure are derived from, cancers including but not limited to primary or metastatic melanoma, 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, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, esophageal cancer, gastric cancer, unresectable ovarian cancer with relapsed or refractory disease.

[0267] The term “transfected” or “transformed” or “transduced,” as used herein, 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.

[0268] The term “specifically binds,” as used herein, refers to an antibody, an antibody fragment or a specific ligand, which recognizes and binds a cognate binding partner (e.g., CD70 or mesothelin (MSLN)) present in a sample, but which does not necessarily and substantially recognize or bind other molecules in the sample.

[0269] The term “functional disruption,” as used herein, refers to a physical or biochemical change to a specific (e.g. , target) nucleic acid (e.g. , gene, RNA transcript, of protein encoded thereby) that prevents its normal expression and/or behavior in the cell. In one embodiment, a functional disruption refers to a modification of the gene via a gene editing method. In one embodiment, a functional disruption prevents expression of a target gene (e.g. , an endogenous gene).

[0270] As used herein, the term “meganuclease” refers to an endonuclease that binds double -stranded DNA at a recognition sequence that is greater than 12 base pairs. In some embodiments, the recognition sequence for a meganuclease of the present disclosure is 22 base pairs. In some embodiments, a meganuclease may be an endonuclease that is derived from I-Crel and may refer to an engineered variant of I-Crel that has been modified relative to natural I-Crel with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties. Methods for producing such modified variants of I-Crel are known in the art (e.g., WO 2007/047859). In some embodiments, a meganuclease binds to double -stranded DNA as a heterodimer or as a “single-chain meganuclease” in which a pair of DNA-binding domains are joined into a single polypeptide using a peptide linker. The term “homing endonuclease” is synonymous with the term “meganuclease.” In some embodiments, meganucleases are substantially non-toxic when expressed in cells, particularly in human T cells, such that cells may be transfected and maintained at 37°C without observing deleterious effects on cell viability or significant reductions in meganuclease cleavage activity when measured using the methods described herein.

[0271] As used herein, the term “single-chain meganuclease” refers to a polypeptide comprising a pair of nuclease subunits joined by a linker. A single-chain meganuclease has the organization: N- terminal subunit - Linker - C-terminal subunit. In some embodiments, the two meganuclease subunits may generally be non-identical in amino acid sequence and may recognize non-identical DNA sequences. Thus, in some embodiments, single-chain meganucleases typically cleave pseudo- palindromic or non-palindromic recognition sequences. In some embodiments, a single-chain meganuclease may be referred to as a “single-chain heterodimer” or “single-chain heterodimeric meganuclease” although it is not, in fact, dimeric. For clarity, unless otherwise specified, the term “meganuclease” can refer to a dimeric or single-chain meganuclease.

[0272] As used herein, the term “TALEN” refers to an endonuclease comprising a DNA-binding domain comprising 16-22 TAL domain repeats fused to any portion of the Fokl nuclease domain. [0273] As used herein, the term “Compact TALEN” refers to an endonuclease comprising a DNA- binding domain with 16-22 TAL domain repeats fused in any orientation to any catalytically active portion of nuclease domain of the I-Tevl homing endonuclease.

[0274] As used herein, the term “CRISPR” refers to a caspase-based endonuclease comprising a caspase, such as Cas9, and a guide RNA that directs DNA cleavage of the caspase by hybridizing to a recognition site in the genomic DNA.

[0275] As used herein, the term “megaTAL” refers to a single-chain nuclease comprising a transcription activator-like effector (TALE) DNA binding domain with an engineered, sequencespecific homing endonuclease.

[0276] As is used herein, the terms “T cell receptor” and “T cell receptor complex” are used interchangeably to refer to a molecule found on the surface of T cells that is, in general, responsible for recognizing antigens. The TCR comprises a heterodimer consisting of a TCR alpha and TCR beta chain in 95% of T cells, whereas 5% of T cells have TCRs consisting of TCR gamma and TCR delta chains. The TCR further comprises one or more of CD3a, CD3y, and CD38. In some embodiments, the TCR comprises CD3a. In some embodiments, the TCR comprises CD3y. In some embodiments, the TCR comprises CD35. In some embodiments, the TCR comprises CD3^. Engagement of the TCR with antigen, e.g., with antigen and MHC, results in activation of its T cells through a series of biochemical events mediated by associated enzymes, co-receptors, and specialized accessory molecules. In some embodiments, the constant domain of TCR alpha has a sequence of SEQ ID NO:711. In some embodiments, the constant domain of TCR alpha has an IgC domain having a sequence of SEQ ID NO:712, a transmembrane domain having a sequence of SEQ ID NO:713, and an intracellular domain having a sequence of SS. In some embodiments, the constant domain of TCR beta has a sequence of SEQ ID NO:715. In some embodiments, the constant domain of TCR beta has an IgC domain having a sequence of SEQ ID NO:716, a transmembrane domain having a sequence of SEQ ID NO:717, and an intracellular domain having a sequence of SEQ ID NO:719. In some embodiments, the constant domain of TCR delta has a sequence of SEQ ID NO:725. In some embodiments, the constant domain of TCR delta has an IgC domain having a sequence of SEQ ID NO:726, a transmembrane domain having a sequence of SEQ ID NO:727, and an intracellular domain having a sequence of L. In some embodiments, the constant domain of TCR gamma has a sequence of SEQ ID NO:721 . In some embodiments, the constant domain of TCR gamma has an IgC domain having a sequence of SEQ ID NO:722, a transmembrane domain having a sequence of SEQ ID NO:723, and an intracellular domain having a sequence of SEQ ID NO:724. In some embodiments, CD3 epsilon has a sequence of SEQ ID NO:694. In some embodiments, CD3 epsilon has an extracellular domain having a sequence of SEQ ID NO:696, a transmembrane domain having a sequence of SEQ ID NO:697, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO:698. In some embodiments, CD3 delta has a sequence of SEQ ID NO:704. In some embodiments, CD3 delta has an extracellular domain having a sequence of SEQ ID NO:706, a transmembrane domain having a sequence of SEQ ID NO:707, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO:708. In some embodiments, CD3 gamma has a sequence of SEQ ID NO:699. In some embodiments, CD3 gamma has an extracellular domain having a sequence of SEQ ID NO:701, a transmembrane domain having a sequence of SEQ ID NO:702, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO:703.

[0277] Ranges: throughout this disclosure, various aspects of the present disclosure 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 present disclosure. 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.

[0278] Provided herein are compositions of matter and methods of use for the treatment of a disease such as cancer, using the composition as provided herein. 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. The term “TRuC™” or T Cell Receptor fusion construct (emphasis added) may also be used interchangeably to describe a TFP. 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, e.g., a single domain antibody or scFv, a transmembrane domain, and cytoplasmic signaling domains (also referred to herein as “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 4-1BB (i.e., CD137), CD27 and/or CD28.

T cell receptor (TCR) fusion proteins (TFP)

[0279] The present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD70, , e.g., human CD70, 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. The present disclosure also encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to mesothelin (MSLN), e.g., human MSLN, 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. The present disclosure further encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD70, , e.g., human CD70, and/or mesothelin (MSLN), e.g., human MSLN, 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. 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.

[0280] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain.

[0281] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain and a TCR epsilon chain.

[0282] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a TCR alpha chain.

[0283] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a TCR beta chain.

[0284] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a TCR gamma chain.

[0285] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a TCR delta chain. [0286] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a CD3 gamma chain.

[0287] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a CD3 delta chain.

[0288] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a CD3 epsilon chain.

[0289] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a TCR alpha chain.

[0290] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a TCR beta chain.

[0291] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a TCR gamma chain.

[0292] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a TCR delta chain.

[0293] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a CD3 gamma chain.

[0294] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a CD3 delta chain.

[0295] In some embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a CD3 epsilon chain.

[0296] In some embodiments, the first TFP, the second TFP, or both incorporate into a TCR or functionally interact with a TCR when expressed in a T cell.

[0297] In some embodiments, the first TFP, the second TFP, or both incorporate into a TCR or functionally interact with a TCR when expressed in a T cell.

[0298] In some embodiments, the encoded first antigen binding domain is connected to the TCR extracellular domain of the first TFP by a first linker sequence, the encoded second antigen binding domain is connected to the TCR extracellular domain of the second TFP by a second linker sequence, or both the first antigen binding domain is connected to the TCR extracellular domain of the first TFP by the first linker sequence and the encoded second antigen binding domain is connected to the TCR extracellular domain of the second TFP by the second linker sequence.

[0299] In some embodiments, the first linker sequence and the second linker sequence comprise (G4S)n, wherein n=l to 4.

[0300] In some embodiments, the TCR subunit of the first TFP, the TCR subunit of the second TFP, or both comprise a TCR extracellular domain.

[0301] In some embodiments, the TCR subunit of the first TFP, the TCR subunit of the second TFP, or both comprise a TCR transmembrane domain.

[0302] In some embodiments, the TCR subunit of the first TFP, the TCR subunit of the second TFP, or both comprise a TCR intracellular domain. [0303] In some embodiments, the TCR subunit of the first TFP, the TCR subunit of the second TFP, or both comprise (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.

[0304] In some embodiments, the TCR subunit of the first TFP, the TCR subunit of the second TFP, or both comprise 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.

[0305] In some embodiments, the TCR subunit of the first TFP, the TCR subunit of the second TFP, or both comprise an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of 4- IBB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.

[0306] In some embodiments, the first human or humanized antibody domain, the second human or humanized antibody domain, or both comprise an antibody fragment.

[0307] In some embodiments, the first human or humanized antibody domain, the second human or humanized antibody domain, or both comprise a scFv or a VH domain.

[0308] In some embodiments, the composition encodes (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 of a light chain binding domain amino acid sequence with 70-100% sequence identity to a light chain sequence of Table 1, and/or (ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of a heavy chain sequence of Table 1.

[0309] In some embodiments, the composition 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 Table 1, or a sequence with 95- 99% identity to a light chain variable region amino acid sequence of Table 1.

[0310] In some embodiments, the composition 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 Table 1, or a sequence with 95-99% identity to a heavy chain variable region amino acid sequence of Table 1.

[0311] In some embodiments, the encoded first TFP, the encoded second TFP, or both include 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.

[0312] In some embodiments, the encoded first TFP and the encoded second TFP include 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.

[0313] In some embodiments, the encoded first TFP and the encoded second TFP include 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, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0314] In some embodiments, the composition further comprises a sequence encoding a costimulatory domain.

[0315] In some embodiments, the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA- 1 (CDl la/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.

[0316] In some embodiments, the composition further comprises comprising a sequence encoding an intracellular signaling domain

[0317] In some embodiments, the composition further comprises a leader sequence.

[0318] In some embodiments, the composition further comprises a protease cleavage site.

[0319] 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 first TFP, the second TFP, or both.

[0320] In some embodiments, the isolated nucleic acid molecule is an mRNA.

[0321] In some embodiments, the first TFP, the second TFP, or both include 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, CD16a, CD16b, 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.

[0322] In some embodiments, the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon.

[0323] 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.

[0324] In some embodiments, the composition further comprises a leader sequence.

[0325] In one aspect, provided herein is a composition comprising a polypeptide molecule encoded by the nucleic acid molecule of a composition described herein.

[0326] In some embodiments, the polypeptide comprises a first polypeptide encoded by a first nucleic acid molecule and a second polypeptide encoded by a second nucleic acid molecule.

[0327] In one aspect, provided herein is a composition comprising a recombinant TFP molecule encoded by the nucleic acid molecule of a composition described herein.

[0328] In one aspect, provided herein is a composition comprising a vector comprising a nucleic acid molecule encoding a polypeptide or recombinant TFP molecule described herein.

[0329] In some embodiments, the vector comprises a) a first vector comprising a first nucleic acid molecule encoding the first TFP; and b) a second vector comprising a second nucleic acid molecule encoding the second TFP.

[0330] In some embodiments, the vector is selected from the group consisting of a DNA, an RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector.

[0331] In some embodiments, the vector further comprises a promoter.

[0332] In some embodiments, the vector is an in vitro transcribed vector.

[0333] In some embodiments, the nucleic acid molecule in the vector further encodes a poly(A) tail.

[0334] In some embodiments, the nucleic acid molecule in the vector further encodes a 3’UTR.

[0335] In some embodiments, the nucleic acid molecule in the vector further encodes a protease cleavage site.

[0336] In one aspect, provided herein is a composition comprising a cell comprising a composition described herein.

[0337] In some embodiments, the cell is a human T cell.

[0338] In some embodiments, the T cell is a CD8+ or CD4+ T cell.

[0339] In some embodiments, the composition 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.

[0340] In some embodiments, the inhibitory molecule comprises a first polypeptide that comprises at least a portion of PD 1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.

[0341] In one aspect, provided herein is a method of treating a mammal having a disease associated with expression of MSLN or CD70 comprising administering to the mammal an effective amount of a composition described herein.

[0342] In some embodiments, the disease associated with CD70 or MSLN, expression is selected from the group consisting of a proliferative disease, a cancer, a malignancy, myelodysplasia, a myelodysplastic syndrome, a preleukemia, a non-cancer related indication associated with expression of CD70, a non-cancer related indication associated with expression of MSLN, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, esophageal cancer, gastric cancer and unresectable ovarian cancer with relapsed or refractory disease. [0343] In some embodiments, the disease is a hematologic cancer selected from the group consisting of B-cell acute lymphoid leukemia (B-ALL), T cell acute lymphoid leukemia (T-ALL), acute lymphoblastic leukemia (ALL); chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell-follicular lymphoma, large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin’s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, preleukemia, a disease associated with CD70 or MSLN expression, and combinations thereof.

[0344] In some embodiments, the cells expressing a first TFP molecule and a second TFP molecule are administered in combination with an agent that increases the efficacy of a cell expressing the first TFP molecule and the second TFP molecule.

[0345] In some embodiments, less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing: an anti-MSLN chimeric antigen receptor (CAR); an anti-CD70 CAR; an anti-MSLN CAR and an anti-CD70 CAR; or a combination thereof. [0346] In some embodiments, the cells expressing the first TFP molecule and a second TFP molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing the first TFP molecule and the second TFP molecule.

[0347] In some embodiments, the cells expressing the first TFP molecule and a second TFP molecule are administered in combination with an agent that treats the disease associated with MSLN or CD70. [0348] In one aspect, described herein are isolated nucleic acid molecules encoding a T cell Receptor (TCR) fusion protein (TFP) that comprise a TCR subunit and a human or humanized antibody domain comprising an anti -tumor antigen binding domain, such as anti-BCMA, anti-CD19, anti CD20, anti- CD22, anti-MUC16, anti-MSLN, anti-CD70, etc. 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 4- IBB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one, two or three modifications thereto.

[0349] In some embodiments, the human or humanized antibody domain comprises an antibody fragment. In some embodiments, the human or humanized antibody domain comprises a scFv or a VH domain.

[0350] 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.

[0351] 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.

[0352] 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.

[0353] In some 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 or zeta chain of the TCR or CD3 epsilon, CD3 gamma and CD3 delta CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, 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.

[0354] In some embodiments, the encoded anti-tumor-associated antigen binding domain is connected to the TCR extracellular domain by a linker sequence. In some instances, the encoded linker sequence comprises (G4S) _n , wherein n=l to 4. In some instances, the encoded linker sequence comprises (G4S) _n , wherein n=2 to 4. In some instances, the encoded linker sequence comprises (G4S) _n , wherein n=l to 3. In some instances, the encoded linker sequence comprises (G4S) _n , wherein n=2 to 5. In some instances, the encoded linker sequence comprises (G4S) _n , wherein n=l to 4.

[0355] In some embodiments, the isolated nucleic acid molecules further comprise 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 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la/CD18), ICOS (CD278), and 4-1BB (CD137), or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto.

[0356] In some embodiments, the isolated nucleic acid molecules further comprise a leader sequence. [0357] Also provided herein are isolated polypeptide molecules encoded by any of the previously described nucleic acid molecules.

[0358] Also provided herein in another aspect, are isolated T cell receptor fusion protein (TFP) molecules that comprise a human or humanized 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 a human or humanized anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain.

[0359] In some embodiments, the anti-tumor-associated antigen binding domain is a scFv or a VH domain. 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.

[0360] 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 (G4S) _n , wherein n=l to 4. In some instances, the linker sequence comprises (G4S) _n , wherein n=2 to 4. In some instances, the linker sequence comprises (G4S) _n , wherein n=l to 3. In some instances, the linker sequence comprises (G4S) _n , wherein n=l to 4. In some instances, the linker sequence comprises (G4S)n, wherein n=2 to 4.

[0361] 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.

[0362] 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. [0363] 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+ 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. [0364] In another aspect, provided herein are isolated TFP molecules that comprise a human or humanized anti-tumor-associated antigen 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.

[0365] In another aspect, provided herein are isolated TFP molecules that comprise a human or humanized anti-tumor-associated antigen 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.

[0366] In another aspect, provided herein are human CD8+ or CD4+ T cells that comprise at least two TFP molecules, the TFP molecules comprising a human or humanized anti-tumor-associated 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+ T cell.

[0367] In another aspect, provided herein are protein complexes that comprise i) a TFP molecule comprising a human or humanized anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and ii) at least one endogenous TCR complex.

[0368] 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 (G4S) _n , wherein n=l to 4. In some instances, the linker sequence comprises (G ₄ S) n, wherein n=2 to 4. In some instances, the linker sequence comprises (G4S) _n , wherein n=l to 3. [0369] Also provided herein are human CD8+ or CD4+ T cells that comprise at least two different TFP proteins per any of the described protein complexes.

[0370] In another aspect, provided herein is a population of human CD8+ or CD4+ T cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules, the TFP molecules comprising a human or humanized anti-tumor-associated 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+ T cell.

[0371] In another aspect, provided herein is a population of human CD8+ or CD4+ T 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.

[0372] In another aspect, provided herein are methods of making a cell comprising transducing a T cell with any of the described vectors.

[0373] 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 a nucleic acid encoding any of the described TFP molecules.

[0374] 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.

[0375] 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. 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 “pre leukemia” 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.

[0376] 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.

[0377] 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. [0378] In one aspect, the TFP of the present disclosure 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 present disclosure include those associated with viral, bacterial and parasitic infections; autoimmune diseases; and cancerous diseases (e.g., malignant diseases).

[0379] 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. [0380] In one aspect, the portion of the TFP comprising the antigen binding domain comprises an antigen binding domain that targets CD70. In one aspect, the antigen binding domain targets human CD70. In one aspect, the portion of the TFP comprising the antigen binding domain comprises an antigen binding domain that targets MSLN. In one aspect, the antigen binding domain targets human MSLN. In one aspect, the portion of the TFP comprising the antigen binding domain comprises an antigen binding domain that targets CD70 and an antigen binding domain that targets MSLN. In one aspect, the antigen binding domains target human CD70 and human MSLN.

[0381] The antigen binding domain can be any domain that binds to the antigen including but not limited to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and 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 (V _L ) 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, anticalin, 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. 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.

[0382] 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 murine, 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 murine, 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 murine, humanized or human anti-CD70 binding domain and/or anti- MSLN binding domain as described herein, e.g. , a murine, humanized or human anti-CD70 binding domain and/or anti- MSLN 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 murine, humanized or human anti-CD70 binding domain and/or anti- MSLN 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 murine, humanized or human anti- TAA binding domain described herein, e.g., the murine, 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 murine, humanized or human anti-TAA binding domain comprises a humanized or human light chain variable region described herein and/or a murine, humanized or human heavy chain variable region described herein. In one embodiment, the murine, humanized or human anti-TAA binding domain comprises a murine, humanized or human heavy chain variable region described herein, e.g., at least two murine, 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 an embodiment, the anti-TAA binding domain (e.g., an scFv or VHH nb) comprises: a light chain variable region as provided herein or 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 as provided herein or 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 murine, humanized or human anti-TAA 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 murine, humanized, or human anti-TAA binding domain includes a (Gly4-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 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. In some instances, the linker region comprises (G4S) _n , wherein n=l to 4. In some instances, the linker region comprises (G4S) _n , wherein n=2 to 5. In some instances, the linker region comprises (G4S) _n , wherein n=l to 5.

[0383] In some embodiments, the antigen binding domain is an antibody or a fragment thereof. In some embodiments, the antigen binding domain is a camelid antibody or a binding fragment thereof. In some embodiments, the antigen binding domain is a murine antibody or a binding fragment thereof. In some embodiments, the antigen binding domain is a human or humanized antibody or a binding fragment thereof. In some embodiments, the antigen binding domain is a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain. In some embodiments, the sdAb is a VHH.

[0384] In some embodiments, the antigen-binding domain comprises an anti-MSLN humanized or human single domain antibody or an antibody fragment having a CDR1 of SEQ ID NO: 60, a CDR2 of SEQ ID NO : 61 , and a CDR3 of SEQ ID NO : 62, a CDR1 of SEQ ID NO : 63 , a CDR2 of SEQ ID NO : 64, and a CDR3 of SEQ ID NO : 65 , or a CDR1 of SEQ ID NO : 66, a CDR2 of SEQ ID NO : 67, and a CDR3 of SEQ ID NO : 68.

[0385] In some embodiments, the antigen-binding domain comprises an anti-MSLN single domain antibody or an antibody fragment comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71. In some embodiments, the antigen-binding domain comprises an anti-MSLN single domain antibody or an antibody fragment having a CDR1, a CDR2, and a CDR3 of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71. In some embodiments, the antigen-binding domain comprises an anti- MSLN single domain antibody or an antibody fragment comprising the sequence of SEQ ID NO:69, SEQ ID NO: 70, or SEQ ID NO:71.

[0386] In some embodiments, the antigen-binding domain comprises an anti-MSLN single domain antibody or an antibody fragment having any one of a CDR1, a CDR2, and a CDR3 sequences listed in Table 1. In some embodiments, the antigen-binding domain comprises an anti-MSLN single domain antibody or an antibody fragment having a CDR1, a CDR2, and a CDR3 of any one of the anti-MSLN single domain antibodies listed in Table 1. In some embodiments, the antigen-binding domain comprises an anti-MSLN single domain antibody or an antibody fragment comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the anti-MSLN single domain antibodies listed in Table 1. In some embodiments, the antigen-binding domain comprises an anti-MSLN single domain antibody or an antibody fragment comprising any one of the anti-MSLN single domain antibodies listed in Table 1. [0387] In some embodiments, the antigen-binding domain comprises an anti- MSLN single chain Fv (scFv) or an antibody fragment thereof. The anti-MSLN scFv or antibody fragment thereof can comprise a heavy chain complementary determining region 1 (CDRH1), a CDRH2, and a CDRH3 sequences listed in Table 1. The anti-MSLN scFv or antibody fragment thereof can comprise a light chain complementary determining region 1 (CDRL1), a CDRL2, and a CDRL3 sequences listed in Table 1. In some embodiments, the antigen-binding domain comprises an anti- MSLN single chain Fv (scFv) or an antibody fragment thereof. The anti-MSLN scFv or antibody fragment thereof can comprise a CDRH1, a CDRH2, and a CDRH3 of any one of the anti-MSLN single domain antibodies listed in Table 1. The anti-MSLN scFv or antibody fragment thereof can comprise a CDRL1, a CDRL2, and a CDRL3 sequences listed in of any one of the anti-MSLN single domain antibodies listed in Table 1. The anti-MSLN scFv or antibody fragment thereof can comprise a heavy chain variable (VH) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the anti-MSLN scFv VH sequences listed in Table 1. The anti-MSLN scFv or antibody fragment thereof can comprise a light chain variable (VL) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the anti-MSLN scFv VL sequences listed in Table 1. The anti-MSLN scFv or antibody fragment thereof can comprise a heavy chain variable (VH) domain having any one of the anti-MSLN scFv VH sequences listed in Table 1. The anti-MSLN scFv or antibody fragment thereof can comprise a light chain variable (VL) domain having any one of the anti-MSLN scFv VL sequences listed in Table 1. [0388] In some embodiments, the antigen-binding domain comprises an anti-CD70 humanized or human single domain antibody or an antibody fragment having a CDR1 of SEQ ID NO: 88, a CDR2 of SEQ ID NO:89, and a CDR3 of SEQ ID NOVO, or a CDR1 of SEQ ID NO:92, a CDR2 of SEQ ID NO : 93 , and a CDR3 of SEQ ID NO : 94, or a CDR1 of SEQ ID NO : 96, a CDR2 of SEQ ID NO : 97, and a CDR3 of SEQ ID NO:98, or a CDR1 of SEQ ID NO: 100, a CDR2 of SEQ ID NO: 101, and a CDR3 of SEQ ID NO: 102, or a CDR1 of SEQ ID NO: 104, a CDR2 of SEQ ID NO: 105, and a CDR3 of SEQ ID NO: 106, or a CDR1 of SEQ ID NO: 108, a CDR2 of SEQ ID NO: 109, and a CDR3 of SEQ ID NO: 110, or a CDR1 of SEQ ID NO: 112, a CDR2 of SEQ ID NO: 113, and a CDR3 of SEQ ID NO: 114, or a CDR1 of SEQ ID NO: 116, a CDR2 of SEQ ID NO: 117, and a CDR3 of SEQ ID NO: 118, or a CDR1 of SEQ ID NO: 120, a CDR2 of SEQ ID NO: 121, and a CDR3 of SEQ ID NO: 122.

[0389] In some embodiments, the anti-CD70 single domain antibody or an antibody fragment comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NOVI, 95, 99, 103, 107, 111, 115, 119, or 123. In some embodiments, the anti-CD70 single domain antibody or an antibody fragment comprises the sequence of SEQ ID NOVI, 95, 99, 103, 107, 111, 115, 119, or 123.

[0390] In some embodiments, the antigen-binding domain comprises an anti-CD70 single chain Fv (scFv) or an antibody fragment thereof. The anti-CD70 scFv or antibody fragment thereof can comprise a heavy chain complementary determining region 1 (CDRH1) having a sequence of SEQ ID NO:361, a CDRH2 having a sequence of SEQ ID NO:362, and a CDRH3 having a sequence of SEQ ID NOs: 363. The anti-CD70 scFv or antibody fragment thereof can comprise a light chain complementary determining region 1 (CDRL1) having a sequence of SEQ ID NO:365 , a CDRL2 having a sequence of SEQ ID NO:366, and a CDRL3 having a sequence of SEQ ID NO:367. The anti-CD70 scFv or antibody fragment thereof can comprise a heavy chain variable (VH) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:364. The anti-CD70 scFv or antibody fragment thereof can comprise a light chain variable (VL) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:368.

[0391] In some embodiments, the antigen-binding domain comprises an anti-CD70 single domain antibody or an antibody fragment having any one of a CDR1, a CDR2, and a CDR3 sequences listed in Table 1. In some embodiments, the antigen-binding domain comprises an anti-CD70 single domain antibody or an antibody fragment having a CDR1, a CDR2, and a CDR3 of any one of the anti-CD70 single domain antibodies listed in Table 1. In some embodiments, the antigen-binding domain comprises an anti-CD70 single domain antibody or an antibody fragment comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the anti-CD70 single domain antibodies listed in Table 1. In some embodiments, the antigen-binding domain comprises an anti-CD70 single domain antibody or an antibody fragment comprising any one of the anti-CD70 single domain antibodies listed in Table 1.

[0392] In some embodiments, the antigen-binding domain comprises an anti- MSLN single chain Fv (scFv) or an antibody fragment thereof. The anti-CD70 scFv or antibody fragment thereof can comprise a heavy chain complementary determining region 1 (CDRH1), a CDRH2, and a CDRH3 sequences listed in Table 1. The anti-CD70 scFv or antibody fragment thereof can comprise a light chain complementary determining region 1 (CDRL1), a CDRL2, and a CDRL3 sequences listed in Table 1. In some embodiments, the antigen-binding domain comprises an anti- MSLN single chain Fv (scFv) or an antibody fragment thereof. The anti-CD70 scFv or antibody fragment thereof can comprise a CDRH1, a CDRH2, and a CDRH3 of any one of the anti-CD70 single domain antibodies listed in Table 1. The anti-CD70 scFv or antibody fragment thereof can comprise a CDRL1, a CDRL2, and a CDRL3 sequences listed in of any one of the anti-CD70 single domain antibodies listed in Table 1. The anti-CD70 scFv or antibody fragment thereof can comprise a heavy chain variable (VH) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the anti-CD70 scFv VH sequences listed in Table 1. The anti-CD70 scFv or antibody fragment thereof can comprise a light chain variable (VL) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the anti-CD70 scFv VL sequences listed in Table 1. The anti-CD70 scFv or antibody fragment thereof can comprise a heavy chain variable (VH) domain having any one of the anti-CD70 scFv VH sequences listed in Table 1. The anti-CD70 scFv or antibody fragment thereof can comprise a light chain variable (VL) domain having any one of the anti-CD70 scFv VL sequences listed in Table 1. [0393] 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. [0394] 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. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 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. US2005/0042664, U.S. Patent Application Publication No. US2005/0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169: 1119-25 (2002), Caldas et al., Protein Eng., 13(5) : 353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., 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.)

[0395] 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 ofWinter 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. Pat. 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 (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., Proc. Natl. Acad. Sci. USA, 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

[0396] 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 al., J. Immunol., 151:2296 (1993); Chothia et al., 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 al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., 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.

[0397] In some aspects, the portion of a TFP composition of the present disclosure 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 present disclosure, 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. [0398] A humanized antibody or antibody fragment may retain a similar antigenic specificity as the original antibody, e.g. , in the present disclosure, the ability to bind human a tumor associated antigen (TAA). In some embodiments, a humanized antibody or antibody fragment may have improved affinity and/or specificity of binding to, e.g., human CD 19, human BCMA, or another tumor associated antigen.

[0399] 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 -TAA 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 present disclosure binds a tumor-associated antigen protein with wild-type or enhanced affinity.

[0400] Also provided herein are methods for obtaining an antibody antigen binding domain specific for a target antigen (e.g., MSLN, CD70, or any target antigen described elsewhere herein for targets of fusion moiety binding domains), 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 VH (or VHH) domain set out herein a VH domain which is an amino acid sequence variant of the VH domain, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH/VI. combination or combinations to identify a specific binding member or an antibody antigen binding domain specific for a target antigen of interest (e.g., MSLN, CD79) and optionally with one or more desired properties.

[0401] In some instances, VH 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 (G4S) _n , wherein n=2 to 4. In some instances, the linker sequence comprises (G4S) _n , wherein n=l to 3. In some instances, the linker region comprises (G4S) _n , wherein n=l to 4. In some instances, the linker region comprises (G4S) _n , wherein n=2 to 5. In some instances, the linker region comprises (G4S) _n , wherein n=l to 5. 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. 2005/0100543, 2005/0175606, 2007/0014794, and PCT publication Nos. W02006/020258 and W02007/024715, each of which is incorporated herein by reference.

[0402] An scFv can comprise a linker of about 10, 11, 12, 13, 14, 15 or greater than 15 residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises amino acids glycine and serine. In another embodiment, the linker sequence comprises sets of glycine and serine repeats such as (Gly4Ser) _n , where n is a positive integer equal to or greater than 1. In one embodiment, the linker can be (Gly4Ser)4 or (Gly4Ser)3. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. In some instances, the linker sequence comprises (G4S) _n , wherein n=2 to 4. In some instances, the linker sequence comprises (G4S) _n , wherein n=l to 3. In some instances, the linker region comprises (G4S) _n , wherein n=l to 4. In some instances, the linker region comprises (G4S) _n , wherein n=2 to 5. In some instances, the linker region comprises (G4S) _n , wherein n=l to 5. In some embodiments, the linker sequence comprises GGSGGSGGSGGS (SEQ ID NO:782).

[0403] The antigen binding domain described herein can be a camelid antibody or binding fragment thereof. The antigen binding domain can be a murine antibody or binding fragment thereof. The antigen binding domain can be a human or humanized antibody or binding fragment thereof. The antigen binding domain can be a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain. In some embodiments, the antigen binding domain is an scFv as listed in Table 1. The antigen binding domain can be a single domain antibody (sdAb). The sdAb can be a VHH. In some embodiments, the antigen binding domain is a VHH listed in Table 1.

Stability and Mutations

[0404] 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.

[0405] The improved thermal stability of the anti-TAA (tumor-associated antigen) binding domain, e.g., scFv is subsequently conferred to the entire TAA-TFP construct, leading to improved therapeutic properties of the anti-TAA TFP construct. The thermal stability of the anti-tumor-associated antigen binding domain, e.g., 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., scFv, 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., scFv, 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 as described 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 in more detail below.

[0406] Mutations in antibody sequences (e.g., arising through humanization or mutagenesis of the soluble scFv or sdAb) alter the stability of the antibody or fragment thereof and improve the overall stability of the antibody and the anti-tumor-associated antigen TFP construct. Stability of the humanized antibody or fragment thereof is compared against the murine antibody or fragment thereof using measurements such as TM, temperature denaturation and temperature aggregation. In one embodiment, the anti-tumor-associated antigen binding domain, e.g., a scFv or sdAb, comprises at least one mutation arising from the humanization process such that the mutated scFv confers improved stability to the anti-TAA TFP construct. In another embodiment, the anti-TAA binding domain, e.g., scFv or sdAb, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations arising from the humanization process such that the mutated scFv or sdAb confers improved stability to the TAA-TFP construct.

[0407] In various aspects, 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 present disclosure comprises an antibody fragment. In a further aspect, that antibody fragment comprises a scFv or sdAb.

[0408] 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 present disclosure comprises an antibody fragment. In a further aspect, that antibody fragment comprises a scFv or sdAb.

[0409] It will be understood by one of ordinary skill in the art that the antibody or antibody fragment of the present disclosure 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.

[0410] 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).

[0411] 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.

[0412] 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’l. 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 al., (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 al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0413] In one aspect, the present disclosure contemplates modifications of the starting antibody or fragment (e.g., scFv or sdAb) 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 or sdAb, 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 antitumor-associated antigen binding domain, e.g., scFv, or the starting sdAb framework region of the anti-tumor-associated antigen binding domain. The present disclosure 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.

Extracellular Domain

[0414] 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 present disclosure 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, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD 154. In some embodiments, the extracellular domain is a TCR extracellular domain. In some instances, the TCR extracellular domain 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 TCR gamma chain, a TCR delta 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.

[0415] In some embodiments, the TCR extracellular domain comprises an extracellular domain or portion thereof of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain. In some embodiments, the TCR extracellular domain comprises the extracellular portion of a constant (an IgC) domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain. [0416] In some embodiments, the extracellular domain comprises, or comprises at least 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, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 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, 100 or more consecutive amino acid residues of the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain. In some embodiments, the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain. In some embodiments, the extracellular domain comprises a sequence encoding the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain having a truncation of 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 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.

[0417] In some embodiments, the extracellular domain comprises, or comprises at least 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, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 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, 100 or more consecutive amino acid residues of the extracellular portion of a constant (an IgC) domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma. In some embodiments, the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular portion of a constant (an IgC) domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma. In some embodiments, the extracellular domain comprises a sequence encoding the extracellular portion of a constant (an IgC) domain of TCR alpha, TCR beta, TCR delta, or TCR gamma having a truncation of 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 or more amino acids at the N- or C-terminus or at both the N- and C- terminus.

[0418] In some embodiments, the extracellular domain comprises, or comprises at least 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, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 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, 100 or more consecutive amino acid residues of the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the extracellular domain comprises a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of 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 or more amino acids at the N- or C-terminus or at both the bland C-terminus.

[0419] The extracellular domain can be a TCR extracellular domain. The TCR extracellular domain can be derived from a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit or a CD3 delta TCR subunit. The extracellular domain can be a full-length TCR extracellular domain or fragment (e.g., functional fragment) thereof. The extracellular domain can comprise a variable domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. The extracellular domain can comprise a variable domain and a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. In some cases, the extracellular domain may not comprise a variable domain.

[0420] The extracellular domain can comprise a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. The extracellular domain can comprise a full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. The extracellular domain can comprise a fragment (e.g, functional fragment) of the full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. For example, the extracellular domain can comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.

[0421] The TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain described herein can be derived from various species. The TCR chain can be a murine or human TCR chain. For example, the extracellular domain can comprise a constant domain of a murine TCR alpha chain, a murine TCR beta chain, a human TCR gamma chain or a human TCR delta chain.

Transmembrane Domain

[0422] 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.

[0423] 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. In some instances, the TCR- integrating subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a TCR zeta chain, a CD3 epsilon, 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, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0424] In some embodiments, the transmembrane domain comprises, or comprises at least 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, or 30 or more consecutive amino acid residues of the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the transmembrane domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the transmembrane domain comprises a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.

[0425] 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.

Linkers

[0426] Optionally, a short oligo- or polypeptide linker, between 2 and 10 amino acids in length may form the linkage between the binding element and the TCR extracellular domain of the TFP. A glycine-serine doublet provides a particularly suitable linker. In some cases, the linker may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more in length. For example, in one aspect, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:690) or a sequence (GGGGS)x wherein X is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more (SEQ ID NO:732). In some embodiments, X is 2. In some embodiments, X is 4. In some embodiments, the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO:691).

Cytoplasmic Domain

[0427] The cytoplasmic domain of the TFP can include an intracellular domain. In some embodiments, the intracellular domain is from CD3 gamma, CD3 delta, CD3 epsilon, TCR alpha, TCR beta, TCR gamma, or TCR delta. In some embodiments, the intracellular domain comprises a signaling domain, if the TFP contains CD3 gamma, delta or epsilon polypeptides; TCR alpha, TCR beta, TCR gamma, and TCR delta subunits generally have short (e.g., 1-19 amino acids in length) intracellular domains and 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. While the intracellular domains of TCR alpha, TCR beta, TCR gamma, and TCR delta do not have signaling domains, the TCR that forms with any TCR fusion protein is able to recruit proteins having a primary intracellular signaling domain described herein, e.g., CD3 zeta, which functions as an intracellular signaling domain. 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.

[0428] Examples of intracellular domains for use in the TFP of the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that are able to 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.

[0429] In some embodiments, the intracellular domain comprises the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.

[0430] In some embodiments, the intracellular domain comprises, or comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more consecutive amino acid residues of the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain. In some embodiments, the intracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain. In some embodiments, the transmembrane domain comprises a sequence encoding the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.

[0431] In some embodiments, the intracellular domain comprises, or comprises at least 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 or more consecutive amino acid residues of the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the intracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the intracellular domain comprises a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of 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 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.

[0432] 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).

[0433] 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).

[0434] Examples of ITAMs containing primary intracellular signaling domains that are of particular use in the present disclosure 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 present disclosure comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3 epsilon, CD3 delta, or CD3 gamma. 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. [0435] The intracellular signaling domain of the TFP can comprise a CD3 signaling domain, e.g., CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta, by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a TFP of the present disclosure. 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-1 (LFA-1), 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).

[0436] The intracellular signaling sequences within the cytoplasmic portion of the TFP of the present disclosure 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.

[0437] In one 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.

[0438] 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., MSLN, or CD70) or a different target (e.g., Nectin-4, CD19, or MUC16). 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 form an association with the antigen binding domain of the second TFP, e.g., the antigen binding domain of the second TFP is a VHH.

[0439] In another aspect, the TFP-expressing cell described herein can further express another agent, e.g., an agent which enhances the activity of a modified T cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., PD1, can, in some embodiments, decrease the ability of a modified T 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 which 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 PD 1 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-1 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. [0440] 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 4 IBB and CD3 zeta (also referred to herein as a PD1 TFP). In one embodiment, the PD 1 TFP, when used in combinations with the TFP as 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).

[0441] In another aspect, the present disclosure 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-CD70 binding domain and/or an anti- MSLN binding domain described herein, and a second cell expressing a TFP having a binding domain specifically targeting a different antigen, e.g., a binding domain described herein that differs from the anti-CD70 binding domain and/or the anti- MSLN 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 a first binding domain 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 the binding domain of the first cell (e.g., another tumor-associated antigen).

[0442] In another aspect, the present disclosure provides a population of cells wherein at least one cell in the population expresses a TFP having a domain described herein, and a second cell expressing another agent, e.g., an agent which enhances the activity of a modified T 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 modified T cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, 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 some embodiments, the agent is a cytokine. In some embodiments, the cytokine is IL-15. In some embodiments, IL- 15 increases the persistence of the T cells described herein.

[0443] TFP constructs can be generated as previously described. An anti -MSLN or CD 19 binder can be linked to a CD3 or TCR DNA fragment by either a DNA sequence encoding a short linker (SL): AAAGGGGSGGGGSGGGGSLE (SEQ ID NO:387) or a long linker (LL): AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE (SEQ ID NO:388) into pLRPO or p510 vector. In some embodiments, the TFP used is TC-210 (e.g., an anti-MSLN MHle VHH antibody linked to CD3 epsilon) having the sequence of SEQ ID NO: 195. In some embodiments, the TFP used is TC-110 (e.g., an anti-CD19 FMC63 scFv antibody linked to CD3 epsilon) having the sequence of SEQ ID NO: 196.

[0444] Anti-MSLN-CD3 epsilon (SEQ ID NO: 195)

MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGGDWSANFMY WYRQ APGKQRELVARISGRGVVDYVESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVAS Y WGQGTLVTVSSAAAGGGGSGGGGSGGGGSLEDGNEEMGGITQTPYKVSISGTTVILTCPQ YP GSE1LWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLY LR ARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQ NKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

Exemplary T cell receptor (TCR) fusion protein (TFP) Constructs

[0445] Exemplary dual T cell receptor (TCR) fusion protein (TFP) constructs are shown in Table 2. [0446] In some embodiments, the first TFP comprises a GM-CSF signal peptide, an anti-CD70 light chain variable region, a whitlow linker, an anti-CD70 heavy chain variable region, an A3(G4S)3LE Linker, and CD3-epsilon polypeptide sequence.

[0447] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, a GM-CSF signal peptide operatively linked to an anti-CD70 light chain variable region operatively linked to a whitlow linker operatively linked to an anti-CD70 heavy chain variable region operatively linked to an A3(G4S)3LE Linker operatively linked to CD3-epsilon polypeptide sequence.

[0448] In some embodiments, the first TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:368, the sequence of SEQ ID NO: 1021, the sequence of SEQ ID NO:364, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:733.

[0449] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO:364 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:733.

[0450] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1038, the sequence of SEQ ID NO: 1042, the sequence of SEQ ID NO: 1043, the sequence of SEQ ID NO: 1044, the sequence of SEQ ID NO: 1045, and the sequence of SEQ ID NO: 1046.

[0451] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046.

[0452] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1049, the sequence of SEQ ID NO: 1042, the sequence of SEQ ID NO: 1043, the sequence of SEQ ID NO: 1044, the sequence of SEQ ID NO: 1045, and the sequence of SEQ ID NO: 1046.

[0453] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046.

[0454] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1060, the sequence of SEQ ID NO: 1061, the sequence of SEQ ID NO: 1062, the sequence of SEQ ID NO: 1063, the sequence of SEQ ID NO: 1064, and the sequence of SEQ ID NO: 1065.

[0455] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1060 operatively linked to the sequence of SEQ ID NO: 1061 operatively linked to the sequence of SEQ ID NO: 1062 operatively linked to the sequence of SEQ ID NO: 1063 operatively linked to the sequence of SEQ ID NO: 1064 operatively linked to the sequence of SEQ ID NO: 1065.

[0456] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1072, the sequence of SEQ ID NO: 1073, the sequence of SEQ ID NO: 1074, the sequence of SEQ ID NO: 1075, the sequence of SEQ ID NO: 1076, and the sequence of SEQ ID NO: 1077.

[0457] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1072 operatively linked to the sequence of SEQ ID NO: 1073 operatively linked to the sequence of SEQ ID NO: 1074 operatively linked to the sequence of SEQ ID NO: 1075 operatively linked to the sequence of SEQ ID NO: 1076 operatively linked to the sequence of SEQ ID NO: 1077.

[0458] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1038, the sequence of SEQ ID NO: 1042, the sequence of SEQ ID NO: 1043, the sequence of SEQ ID NO: 1044, the sequence of SEQ ID NO: 1040, and the sequence of SEQ ID NO: 1041.

[0459] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1040 operatively linked to the sequence of SEQ ID NO: 1041.

[0460] In some embodiments, the first TFP comprises a GM-CSF signal peptide, an anti-CD70 light chain variable region, a whitlow linker, an anti-CD70 heavy chain variable region, an A3(G4S)3LE Linker, and CD3-gamma polypeptide sequence.

[0461] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, a GM-CSF signal peptide operatively linked to an anti-CD70 light chain variable region operatively linked to a whitlow linker operatively linked to an anti-CD70 heavy chain variable region operatively linked to an A3(G4S)3LE Linker operatively linked to CD3-gamma polypeptide sequence.

[0462] In some embodiments, the first TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:368, the sequence of SEQ ID NO: 1021, the sequence of SEQ ID NO:364, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:734.

[0463] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO:364 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:734.

[0464] In some embodiments, the second TFP comprises a GM-CSF signal peptide, an anti-MSLN sdAb, an A3(G4S)3LE Linker, and CD3-gamma polypeptide.

[0465] In some embodiments, the second TFP comprises, from the N-terminus to the C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN sdAb operatively linked to an A3(G4S)3LE Linker operatively linked to CD3-gamma polypeptide.

[0466] In some embodiments, the second TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:69, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:734. [0467] In some embodiments, the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:69 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:734.

[0468] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1038, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1040, and the sequence of SEQ ID NO: 1041.

[0469] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1040 operatively linked to the sequence of SEQ ID NO: 1041.

[0470] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1049, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1050, and the sequence of SEQ ID NO: 1041.

[0471] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1050 operatively linked to the sequence of SEQ ID NO: 1041.

[0472] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1049, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1045, and the sequence of SEQ ID NO: 1041.

[0473] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1041.

[0474] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1055, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1050, and the sequence of SEQ ID NO: 1041.

[0475] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1055 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1050 operatively linked to the sequence of SEQ ID NO: 1041.

[0476] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1068, the sequence of SEQ ID NO: 1069, the sequence of SEQ ID NO: 1070, and the sequence of SEQ ID NO: 1071.

[0477] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1068 operatively linked to the sequence of SEQ ID NO: 1069 operatively linked to the sequence of SEQ ID NO: 1070 operatively linked to the sequence of SEQ ID NO: 1071.

[0478] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1080, the sequence of SEQ ID NO: 1081, the sequence of SEQ ID NO: 1082, and the sequence of SEQ ID NO: 1083.

[0479] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1080 operatively linked to the sequence of SEQ ID NO: 1081 operatively linked to the sequence of SEQ ID NO: 1082 operatively linked to the sequence of SEQ ID NO: 1083.

[0480] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1085, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1086, and the sequence of SEQ ID NO: 1046.

[0481] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1085 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1086 operatively linked to the sequence of SEQ ID NO: 1046.

[0482] In some embodiments, the second TFP comprises a GM-CSF signal peptide, an anti-MSLN sdAb, an A3(G4S)3LE Linker, and CD3-epsilon polypeptide.

[0483] In some embodiments, the second TFP comprises, from the N-terminus to the C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN sdAb operatively linked to an A3(G4S)3LE Linker operatively linked to CD3-epsilon polypeptide.

[0484] In some embodiments, the second TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:69, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO: 1018. [0485] In some embodiments, the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:69 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO: 1018. [0486] In some embodiments, the second TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:70, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:733.

[0487] In some embodiments, the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:70 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:733.

[0488] In some embodiments, the second TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:69, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:733. [0489] In some embodiments, the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:69 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:733.

[0490] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1049, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1050, and the sequence of SEQ ID NO: 1051.

[0491] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1050 operatively linked to the sequence of SEQ ID NO: 1051.

[0492] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1055, the sequence of SEQ ID NO: 1059, the sequence of SEQ ID NO: 1045, and the sequence of SEQ ID NO: 1046.

[0493] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1055 operatively linked to the sequence of SEQ ID NO: 1059 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046.

[0494] In some embodiments, the second TFP comprises a GM-CSF signal peptide, a first anti- MSLN sdAb, a TLGM Linker, a first anti-MSLN sdAb, an A3(G4S)3LE Linker, and CD3-gamma polypeptide.

[0495] In some embodiments, the second TFP comprises, from the N-terminus to the C-terminus, a GM-CSF signal peptide operatively linked to a first anti-MSLN sdAb operatively linked to a TLGM Linker operatively linked to a first anti-MSLN sdAb operatively linked to an A3(G4S)3LE Linker operatively linked to CD3 -gamma polypeptide.

[0496] In some embodiments, the second TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:69, the sequence of SEQ ID NO:248, the sequence of SEQ ID NO:69, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:734.

[0497] In some embodiments, the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:69 operatively linked to the sequence of SEQ ID NO:248 operatively linked to the sequence of SEQ ID NO:69 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:734.

[0498] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1049, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1056, the sequence of SEQ ID NO: 1057, the sequence of SEQ ID NO: 1050, and the sequence of SEQ ID NO: 1041.

[0499] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1056 operatively linked to the sequence of SEQ ID NO: 1057 operatively linked to the sequence of SEQ ID NO: 1050 operatively linked to the sequence of SEQ ID NO: 1041.

[0500] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1055, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1058, the sequence of SEQ ID NO: 1057, the sequence of SEQ ID NO: 1045, and the sequence of SEQ ID NO: 1041.

[0501] In some embodiments, the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1055 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1058 operatively linked to the sequence of SEQ ID NO: 1057 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1041.

[0502] In some embodiments, the first TFP is operatively linked to the second TFP by the sequence of GSG operatively linked to a T2A linker.

[0503] In some embodiments, the first TFP is operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the T2A linker operatively linked to the N-terminus of the second TFP.

[0504] In some embodiments, the second TFP is operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the T2A linker operatively linked to the N-terminus of the first TFP.

[0505] In some embodiments, the T2A linker comprises the sequence of SEQ ID NO:23.

[0506] In some embodiments, the T2A linker is encoded by the nucleotide sequence of SEQ ID NO: 1048.

[0507] In some embodiments, the T2A linker is encoded by the nucleotide sequence of SEQ ID NO: 1067.

[0508] In some embodiments, the T2A linker is encoded by the nucleotide sequence of SEQ ID NO: 1079.

[0509] In some embodiments, the sequence of GSG is encoded by the nucleotide sequence of SEQ ID NO: 1047.

[0510] In some embodiments, the sequence of GSG is encoded by the nucleotide sequence of SEQ ID NO: 1066.

[0511] In some embodiments, the sequence of GSG is encoded by the nucleotide sequence of SEQ ID NO: 1078.

[0512] In some embodiments, the first TFP is operatively linked to the second TFP by a furin cleavage site operatively linked to the sequence of GSG operatively linked to a T2A linker.

[0513] In some embodiments, the first TFP is operatively linked to the N-terminus of the furin cleavage site operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the T2A linker operatively linked to the N-terminus of the second TFP.

[0514] In some embodiments, the second TFP is operatively linked to the N-terminus of the furin cleavage site operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the T2A linker operatively linked to the N-terminus of the first TFP.

[0515] In some embodiments, the furin cleavage site comprises the sequence of SEQ ID NO: 1019.

[0516] In some embodiments, the furin cleavage site is encoded by the nucleotide sequence of SEQ ID NO: 1052.

[0517] In some embodiments, the T2A linker comprises the sequence of SEQ ID NO:23.

[0518] In some embodiments, the T2A linker is encoded by the nucleotide sequence of SEQ ID NO: 1048.

[0519] In some embodiments, the sequence of GSG is encoded by the nucleotide sequence of SEQ ID NO: 1053.

[0520] In some embodiments, the first TFP is operatively linked to the second TFP by the sequence of GSG operatively linked to a P2A linker.

[0521] In some embodiments, the first TFP is operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the P2A linker operatively linked to the N-terminus of the second TFP.

[0522] In some embodiments, the second TFP is operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the P2A linker operatively linked to the N-terminus of the first TFP.

[0523] In some embodiments, the P2A linker comprises the sequence of SEQ ID NO: 1652.

[0524] In some embodiments, the P2A linker is encoded by the nucleotide sequence of SEQ ID NO: 1054.

[0525] In some embodiments, the sequence of GSG is encoded by the nucleotide sequence of SEQ ID NO: 1053.

[0526] In some embodiments, the first TFP is operatively linked to the second TFP by a furin cleavage site operatively linked to the sequence of GSG operatively linked to a P2A linker.

[0527] In some embodiments, the first TFP is operatively linked to the N-terminus of the furin cleavage site operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the P2A linker operatively linked to the N-terminus of the second TFP.

[0528] In some embodiments, the second TFP is operatively linked to the N-terminus of the furin cleavage site operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the P2A linker operatively linked to the N-terminus of the first TFP.

[0529] In some embodiments, the furin cleavage site comprises the sequence of SEQ ID NO: 1019.

[0530] In some embodiments, the furin cleavage site is encoded by the nucleotide sequence of SEQ ID NO: 1052.

[0531] In some embodiments, the P2A linker comprises the sequence of SEQ ID NO: 1652.

[0532] In some embodiments, the P2A linker is encoded by the nucleotide sequence of SEQ ID NO: 1054.

[0533] In some embodiments, the sequence of GSG is encoded by the nucleotide sequence of SEQ ID NO: 1053.

[0534] In some embodiments, the first TFP comprises having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 1002.

[0535] In some embodiments, the first TFP comprises the sequence of SEQ ID NO: 1002.

[0536] In some embodiments, the first TFP is encoded by a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 1023.

[0537] In some embodiments, the first TFP is encoded by a nucleotide sequence having the sequence of SEQ ID NO: 1023.

[0538] In some embodiments, the second TFP comprises having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1001, 1010, and 1011.

[0539] In some embodiments, the second TFP comprises any one of the sequences selected from SEQ ID NOs: 1001, 1010, and 1011.

[0540] In some embodiments, the second TFP is encoded by a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1022, 1031, and 1032.

[0541] In some embodiments, the second TFP is encoded by a nucleotide sequence having any one of the sequences selected from SEQ ID NOs: 1022, 1031, and 1032.

[0542] In some embodiments, the composition comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1003-1009, 1012, 1013, and 1016.

[0543] In some embodiments, the composition comprises any one of the sequences selected from SEQ ID NOs: 1003-1009, 1012, 1013, and 1016.

[0544] In some embodiments, the composition comprises a sequence encoded by a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the nucleotide sequences selected from SEQ ID NOs: 1024-1030, 1033, 1034, and 1037.

[0545] In some embodiments, the composition comprises a sequence encoded by any one of the nucleotide sequences selected from SEQ ID NOs: 1024-1030, 1033, 1034, and 1037.

[0546] In some embodiments, the TFP comprises a GM-CSF signal peptide, an anti-CD70 light chain variable region, a whitlow linker, an anti-CD70 heavy chain variable region, a G4S5 Linker, an anti- MSLN sdAb, an A3(G4S)3LE Linker, and CD3-epsilon polypeptide.

[0547] In some embodiments, the TFP comprises, from the N-terminus to the C-terminus, a GM-CSF signal peptide operatively linked to an anti-CD70 light chain variable region operatively linked to a whitlow linker operatively linked to an anti-CD70 heavy chain variable region operatively linked to a G4S5 Linker operatively linked to an anti-MSLN sdAb operatively linked to an A3(G4S)3LE Linker operatively linked to CD3 -epsilon polypeptide.

[0548] In some embodiments, the TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:368, the sequence of SEQ ID NO: 1021, the sequence of SEQ ID NO:364, the sequence of SEQ ID NO: 1020, the sequence of SEQ ID NO:69, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:733.

[0549] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:69 operatively linked to the sequence of SEQ ID NO: 1020 operatively linked to the sequence of SEQ ID NO:368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO:364 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:733.

[0550] In some embodiments, the composition comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1003-1009, 1012, 1013, and 1016.

[0551] In some embodiments, the composition comprises any one of the sequences selected from SEQ ID NOs: 1003-1009, 1012, 1013, and 1016.

[0552] In some embodiments, the composition comprises a sequence encoded by a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the nucleotide sequences selected from SEQ ID NOs: 1024-1030, 1033, 1034, and 1037.

[0553] In some embodiments, the composition comprises a sequence encoded by any one of the nucleotide sequences selected from SEQ ID NOs: 1024-1030, 1033, 1034, and 1037.

[0554] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1038, the sequence of SEQ ID NO: 1042, the sequence of SEQ ID NO: 1043, the sequence of SEQ ID NO: 1044, the sequence of SEQ ID NO: 1084, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1045, and the sequence of SEQ ID NO: 1046.

[0555] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1055 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1084 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046.

[0556] In some embodiments, the first TFP comprises a GM-CSF signal peptide, an anti-MSLN sdAb, a G4S5 Linker, an anti-CD70 light chain variable region, a whitlow linker, an anti-CD70 heavy chain variable region, an A3(G4S)3LE Linker, and CD3-epsilon polypeptide.

[0557] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN sdAb operatively linked to a G4S5 Linker operatively linked to an anti-CD70 light chain variable region operatively linked to a whitlow linker operatively linked to an anti-CD70 heavy chain variable region operatively linked to an A3(G4S)3LE Linker operatively linked to CD3 -epsilon polypeptide.

[0558] In some embodiments, the first TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:69, the sequence of SEQ ID NO: 1020, the sequence of SEQ ID NO:368, the sequence of SEQ ID NO: 1021, the sequence of SEQ ID NO:364, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO: 733.

[0559] In some embodiments, the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:69 operatively linked to the sequence of SEQ ID NO: 1020 operatively linked to the sequence of SEQ ID NO:368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO:364 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:733.

[0560] In some embodiments, the first TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1055, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1084, the sequence of SEQ ID NO: 1042, the sequence of SEQ ID NO: 1043, the sequence of SEQ ID NO: 1044, the sequence of SEQ ID NO: 1045, and the sequence of SEQ ID NO: 1046.

[0561] In some embodiments, the TFP is encoded by a nucleotide sequence comprising, from 5 ’-end to 3 ’-end, the sequence of SEQ ID NO: 1055 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1084 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046.

[0562] In some embodiments, the first TFP comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 1014 or SEQ ID NO: 1015.

[0563] In some embodiments, the first TFP comprises the sequence of SEQ ID NO: 1014 or SEQ ID NO: 1015.

[0564] In some embodiments, the first TFP is encoded by a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 1035 or SEQ ID NO: 1036.

[0565] In some embodiments, the first TFP is encoded by the sequence of SEQ ID NO: 1035 or SEQ ID NO: 1036.

Nucleic Acid Constructs Encoding a TFP

[0566] The present disclosure 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.

[0567] In some instances, the recombinant nucleic acid further comprises a leader sequence. In some instances, the recombinant nucleic acid further comprises a promoter sequence. In some instances, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some instances, the recombinant nucleic acid further comprises a 3’UTR sequence. In some instances, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. Non-naturally occurring nucleic acids are well known to those of skill in the art. In some instances, the nucleic acid is an in vitro transcribed nucleic acid.

[0568] Disclosed herein are methods for producing in vitro transcribed RNA encoding TFPs. The present disclosure 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.

[0569] In one aspect, the TFP is encoded by a messenger RNA (mRNA). In one aspect the mRNA encoding the 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 disclosure. 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.

[0570] 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.

[0571] 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.

[0572] 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 that can be used to achieve optimal translation efficiency following transfection of the transcribed RNA.

[0573] 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.

[0574] 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. 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.

[0575] 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.

[0576] 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.

[0577] 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)).

[0578] 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.

[0579] 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.

[0580] 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.

[0581] 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 al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7: 1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958- 966 (2005)).

[0582] 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.

[0583] 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)).

[0584] For additional information on making and using TFP T cells, see U.S. Patent Nos. 10,442,849, 10,358,473, 10,358,474, and 10,208,285, each of which is herein incorporated by reference.

[0585] In some cases, the recombinant nucleic acid molecule described herein can further comprise a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain, a TCR alpha constant domain and a TCR beta constant domain, a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain. The TCR subunit and the antigen binding domain (e.g., an anti-CD70 binding domain or antibody domain, and/or an anti- MSLN binding domain or antibody domain) can be operatively linked. The TFP can functionally incorporate into a TCR complex (e.g., an endogenous TCR complex) when expressed in a T cell. The sequence encoding the TFP and the sequence encoding the TCR constant domain can be contained within a same nucleic acid molecule. The sequence encoding the TFP and the sequence encoding the TCR constant domain can be contained within different nucleic acid molecules. The sequence can further encode a cleavage site (e.g., a protease cleavage site) between the encoded TFP and the TCR constant domain. The cleavage site can be a protease cleavage site. The cleavage site can be a self-cleaving peptide such as a T2A, P2A, E2A or F2A cleavage site.

[0586] The constant domain can comprise a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. The constant domain can comprise a full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. The constant domain can comprise a fragment (e.g., functional fragment) of the full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. For example, the constant domain can comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. The sequence encoding the TCR constant domain can further encode the transmembrane domain and/or intracellular region of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. The sequence encoding the TCR constant domain can encode a full-length constant region of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. The constant region of a TCR chain can comprise a constant domain, a transmembrane domain, and an intracellular region. The constant region of a TCR chain can also exclude the transmembrane domain and the intracellular region of the TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.

[0587] The TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain described herein can be derived from various species. The TCR chain can be a murine or human TCR chain. For example, the constant domain can comprise a constant domain of a murine or human TCR alpha chain, TCR beta chain, TCR gamma chain or TCR delta chain.

[0588] The constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein. For example, the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215. For example, the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215. For example, the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215. The constant domain can comprise a sequence or fragment thereof of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215. The constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO : 711 , SEQ ID NO : 715 , SEQ ID NO : 211 , SEQ ID NO : 721 , SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215. The constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215. The constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.

[0589] The constant domain described herein can be a human TCR alpha constant domain. For example, the human TCR alpha constant domain can comprise a sequence of SEQ ID NO:711 . The human TCR alpha constant domain can comprise truncations, additions, or substitutions of a sequence of SEQ ID NO:711 . For example, the human TCR alpha constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:711. For example, the human TCR alpha constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:711. For example, the human TCR alpha constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:711. The human TCR alpha constant domain can comprise a sequence or fragment thereof of SEQ ID NO:711. The constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:711. The human TCR alpha constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:711. The human TCR alpha constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:711.

[0590] The constant domain described herein can be a human TCR beta constant domain. For example, the human TCR beta constant domain can comprise a sequence of SEQ ID NO:715 or SEQ ID NO:211. The human TCR beta constant domain can comprise truncations, additions, or substitutions of a sequence of SEQ ID NO:715 or SEQ ID NO:211. For example, the human TCR beta constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:715 or SEQ ID NO:211. For example, the human TCR beta constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:715 or SEQ ID NO:211 . For example, the human TCR beta constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:715 or SEQ ID NO:211. The human TCR beta constant domain can comprise a sequence or fragment thereof of SEQ ID NO:715 or SEQ ID NO:211. The constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:715 or SEQ ID NO:211. The human TCR beta constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:715 or SEQ ID NO:211. The human TCR beta constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO : 715 or SEQ ID NO : 211.

[0591] The constant domain described herein can be a human TCR gamma constant domain. For example, the human TCR gamma constant domain can comprise a sequence of SEQ ID NO:721. The human TCR gamma constant domain can comprise truncations, additions, or substitutions of a sequence of SEQ ID NO:721. For example, the human TCR gamma constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:721. For example, the human TCR gamma constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:721. For example, the human TCR gamma constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:721. The human TCR gamma constant domain can comprise a sequence or fragment thereof of SEQ ID NO:721. The constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:721. The human TCR gamma constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:721. The human TCR gamma constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:721.

[0592] The constant domain described herein can be a human TCR delta constant domain. For example, the human TCR delta constant domain can comprise a sequence of SEQ ID NO:725. The human TCR delta constant domain can comprise truncations, additions, or substitutions of a sequence of SEQ ID NO:725. For example, the human TCR delta constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:725. For example, the human TCR delta constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:725. For example, the human TCR delta constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:725. The human TCR delta constant domain can comprise a sequence or fragment thereof of SEQ ID NO:725. The constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:725. The human TCR delta constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:725. The human TCR delta constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO: 725.

[0593] The constant domain described herein can be a murine TCR alpha constant domain. For example, the murine TCR alpha constant domain can comprise a sequence of SEQ ID NO:212 or SEQ ID NO:213. The murine TCR alpha constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein. For example, the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:212 or SEQ ID NO:213. For example, the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:212 or SEQ ID NO:213. For example, the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:212 or SEQ ID NO:213. The constant domain can comprise a sequence or fragment thereof of SEQ ID NO:212 or SEQ ID NO:213. The constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:212 or SEQ ID NO:213. The constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:212 or SEQ ID NO:213. The constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:212 or SEQ ID NO:213.

[0594] The constant domain described herein can be a murine TCR beta constant domain. For example, the murine TCR beta constant domain can comprise a sequence of SEQ ID NO:214 or SEQ ID NO:215. The murine TCR beta constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein. For example, the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:214 or SEQ ID NO:215. For example, the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:214 or SEQ ID NO:215. For example, the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:214 or SEQ ID NO:215. The constant domain can comprise a sequence or fragment thereof of SEQ ID NO:214 or SEQ ID NO:215. The constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:214 or SEQ ID NO:215. The constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:214 or SEQ ID NO:215. The constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:214 or SEQ ID NO:215.

Gene Editing of TCR Complex or Endogenous Protein-coding Genes

[0595] 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., U.S. 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 Review s 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).

[0596] 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.

[0597] 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, mentioned endogenous TCR gene encodes a TCR gamma chain, a TCR delta chain, or a TCR gamma chain and a TCR delta 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 techniques 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 B2M, and/or programmed cell death protein 1 (PD1), and/or other genes. [0598] 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.

[0599] 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 basepair. 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-TevI homing endonuclease. Unlike Fokl, I-TevI does not need to dimerize to produce a double-strand DNA break so a Compact TALEN is functional as a monomer.

[0600] Engineered endonucleases based on the CRISPR/Cas9 system are also known in the art (Ran et al. (2013), NatProtoc. 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). [0601] There are two classes of CRISPR systems known in the art (Adli (2018) Nat. Commun. 9: 1911), 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 most widely used CRISPR/Cas system is 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.

[0602] 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 Sei. 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.

[0603] 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.

[0604] 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 I. 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.

Vectors

[0605] In some embodiments, the instant invention provides vectors comprising the recombinant nucleic acid(s) encoding the TFP and/or additional molecules of interest (e.g., a protein or proteins to be secreted by the TFP T cell). In some instances, the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, an adeno-associated viral vector (AAV), a Rous sarcoma viral (RSV) vector, or a retrovirus vector. In some instances, the vector is an AAV6 vector. In some instances, the vector further comprises a promoter. In some instances, the vector is an in vitro transcribed vector.

[0606] 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.

[0607] The present disclosure also provides vectors in which a DNA of the present disclosure is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve longterm 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.

[0608] In another embodiment, the vector comprising the nucleic acid encoding the desired TFP of the present disclosure is an adenoviral vector (A5/35). In another embodiment, the expression of nucleic acids encoding TFPs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See, e.g., June et al., 2009 Nature Reviews Immunology 9.10: 704-716, which is incorporated herein by reference.

[0609] The TFP of the present invention may be used in multicistronic vectors or vectors expressing several proteins in the same transcriptional unit. Such vectors may use internal ribosomal entry sites (IRES). Since IRES are not functional in all hosts and do not allow for the stoichiometric expression of multiple protein, self-cleaving peptides may be used instead. For example, several viral peptides are cleaved during translation and allow for the expression of multiple proteins form a single transcriptional unit. Such peptides include 2A-peptides, or 2A-like sequences, from members of the Picomaviridae virus family. See for example Szymczak et al., 2004, Nature Biotechnology; 22:589- 594. In some embodiments, the recombinant nucleic acid described herein encodes the TFP in frame with the agent, with the two sequences separated by a self-cleaving peptide, such as a 2A sequence, or a T2A sequence.

[0610] The expression constructs of the present disclosure 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. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, each of which is incorporated by reference herein in their entireties). In another embodiment, the present disclosure provides a gene therapy vector.

[0611] 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.

[0612] 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, for example, 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).

[0613] 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.

[0614] 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.

[0615] 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- 1 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 al., 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 present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present disclosure. 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.

[0616] 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.

[0617] 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.

[0618] 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.

[0619] 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, for example, Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection

[0620] 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).

[0621] 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.

[0622] 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.

[0623] 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.

[0624] 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 disclosure, 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 present disclosure.

[0625] The present disclosure 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.

[0626] The TFP-encoding nucleic acid construct as described herein can be cloned into a lentiviral expression vector and expression validated based on the quantity and quality of the effector T cell response of transduced T cells in response to MSLN+ 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).

Circular RNA

[0627] In some embodiments, TFP T cells are transduced with an RNA molecule. In some embodiments, the RNA is circular RNA. In some embodiments, the 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 in vivo or ex vivo.

[0628] 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 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 al., 2015; Szabo et al., 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 al., 2018 for review).

[0629] To generate circular RNAs that we could subsequently transfer into cells, in vitro production of circular RNAs with autocatalytic-splicing introns can be programmed. A method for generating circular RNA can involve 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 preferred embodiments, precursor RNA was 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 one aspect, the template includes sequences for the TFP, CAR, and TCR, or combination thereof.

[0630] The group I intron of phage T4 thymidylate synthase (td) gene is well characterized to circularize while the exons linearly splice together (Chandry and Bel- fort, 1987; Ford and Ares, 1994; Perriman and Ares, 1998). When the td intron order is permuted flanking any exon sequence, the exon is circularized via two autocatalytic transesterification reactions (Ford and Ares, 1994; Puttaraju and Been, 1995). In preferred embodiments, the group I intron of phage T4 thymidylate synthase (td) gene is used to generate exogenous circular RNA.

[0631] In some exemplary embodiments, a ribozymatic method utilizing a permuted group I catalytic intron has been used since it is more applicable to long RNA circularization and requires only the addition of GTP and Mg ²⁺ as cofactors. This permuted intron-exon (PIE) splicing strategy consists of fused partial exons flanked by half-intron sequences. In vitro, these constructs undergo the double transesterification reactions characteristic of group I catalytic introns, but because the exons are fused, they are excised as covalently 5' to 3' linked circles.

[0632] In one aspect, disclosed herein is a sequence containing a full-length encephalomyocarditis virus (such as EMCV) IRES, a gene encoding a TFP, a CAR, a TCR or combination thereof, two short regions corresponding to exon fragments (El and E2), and of the PIE construct between the 3' and 5' introns of the permuted group I catalytic intron in the thymidylate synthase (Td) gene of the T4 phage or the permuted group I catalytic intron in the pre-tRNA gene of Anabaena. In more preferred embodiments, the mentioned sequence further comprises complementary ‘homology arms’ placed at the 5' and 3' ends of the precursor RNA with the aim of bringing the 5' and 3' splice sites into proximity of one another. To ensure that the major splicing product was circular, the splicing reaction can be treated with RNase R.

[0633] In one aspect, the TFP as described herein is encoded by a circular RNA. In one aspect the circular RNA encoding the TFP as described herein 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.

[0634] In some aspects, linear precursor RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template as is described herein.

[0635] For additional information on TFP T cells produced by the methods above, see copending Provisional Application Serial No. 62/836,977, which is herein incorporated by reference.

Modified T cells

[0636] Disclosed herein are modified T cells comprising the nucleic acid encoding the TFP of the nucleic acid disclosed herein or a TFP encoded by the nucleic acid disclosed herein. Further disclosed herein, in some embodiments, are modified allogenic T cells comprising the nucleic acid TFP disclosed herein or a TFP encoded by nucleic acid disclosed herein.

[0637] In some embodiments, the modified T cells comprising the recombinant nucleic acid disclosed herein, or the vectors disclosed herein comprises a functional disruption of an endogenous TCR. Further disclosed herein, in some embodiments, are modified allogenic T cells comprising the nucleic acid encoding the TFP disclosed herein or a TFP encoded by the nucleic acid disclosed herein. [0638] 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 T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain. In some instances, the endogenous TCR that is functionally disrupted is an endogenous TCR gamma chain, an endogenous TCR delta chain, or an endogenous TCR gamma chain and an endogenous TCR delta 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. In some instances, the T cell is an allogenic T cell. In some instances, the T cell is a TCR alpha-beta T cell. In some instances, the T cell is a TCR gamma-delta T cell. In some instances, one or more of TCR alpha, TCR beta, TCR gamma, and TCR delta have been modified to produce an allogeneic T cell. See, e.g., copending PCT Publication No. WO2019173693, which is herein incorporated by reference.

[0639] In some embodiments, the modified T cells are y5 T cells and do not comprise a functional disruption of an endogenous TCR. In some embodiments, the y5 T cells are V51+ V52- yd T cells. In some embodiments, the y5 T cells are V51- V 52+ yd T cells. In some embodiments, the y5 T cells are V51- N 62- y5 T cells. In some embodiments, the cell is a human NKT cell. In some embodiments, the cell is an allogeneic cell or an autologous cell. In some embodiments, the T cell is modified to comprise a functional disruption of the TCR. In some embodiments, the modified T cells are T cells and do not comprise a functional disruption of an endogenous TCR. 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 CD8+ T cell, a CD4+ T cell, a naive T cell, a memory stem T cell, a central memory T cell, a double negative T cell, an effector memory T cell, an effector T cell, a ThO cell, a TcO cell, a Thl cell, a Tel cell, a Th2 cell, a Tc2 cell, a Th 17 cell, a Th22 cell, a gamma delta T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a hematopoietic stem cell, or a pluripotent stem cell.

[0640] The present disclosure provides genetically-modified immune cells and populations thereof and methods for producing the same. In some embodiments, the genetically-modified immune cells of the presently disclosed compositions and methods are human immune cells. In some embodiments, the immune cells are T cells, or cells derived therefrom. In other embodiments, the immune cells are natural killer (NK) cells, or cells derived therefrom. In still other embodiments, the immune cells are B cells, or cells derived therefrom. In yet other embodiments, the immune cells are monocyte or macrophage cells or cells derived therefrom.

[0641] As used herein, detectable cell-surface expression of an endogenous TCR refers to the ability to detect one or more components of the TCR complex (e.g., an alpha/beta TCR complex) on the cell surface of an immune cell using standard experimental methods. Such methods can include, for example, immunostaining and/or flow cytometry specific for components of the TCR itself, such as a TCR alpha or TCR beta chain, or for components of the assembled cell-surface TCR complex, such as CD3. Methods for detecting cell-surface expression of an endogenous TCR (e.g., an alpha/beta TCR) on an immune cell include those described in the examples herein, and, for example, those described in MacLeod et al. (2017) Molecular Therapy 25(4): 949-961.

[0642] 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.

[0643] The modified T cells can further comprise an enhancing agent or a nucleic acid sequence encoding an enhancing agent. For example, the modified T cells can comprise a nucleic acid sequence encoding a TFP described herein and an additional nucleic acid sequence encoding the enhancing agent. The nucleic acid sequence encoding the TFP and the additional nucleic acid sequence encoding the enhancing agent can be on the same nucleic acid molecule or be on different nucleic acid molecules. In some cases, , the nucleic acid sequence encoding the TFP and the additional nucleic acid sequence encoding the enhancing agent are operatively linked by a linker. For example, the linker may be a cleavable linker. In some embodiments, the linker may comprise a protease cleavage site. The cleavage site can be a self-cleaving peptide, for example, a 2A cleavage site such as a T2A, P2A, E2A or F2A cleavage site. In some embodiments, the protease cleavage site is a T2A cleavage site. The enhancing agent can be a PD-1 switch molecule comprising a PD-1 polypeptide and an intracellular domain of a costimulatory polypeptide, an anti -PD-1 antibody, or a fusion molecule comprising an anti -PD-1 antibody or fragment thereof and a transmembrane domain.

PD-1 Switch Molecules

[0644] The modified cells can comprise 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. The modified cells can comprise a nucleic acid sequence 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 embodiments, the inhibitory molecule can be a PD-1 switch molecule comprising the first polypeptide comprising at least a portion of PD-1 and the second polypeptide comprising a costimulatory domain and primary signaling domain. In some embodiments, a T cell expressing the TFP as described herein, and a PD-1 switch molecule as descried herein can inhibit tumor growth when expressed in a T cell. The PD-1 switch molecule can enhance the activity of a modified T cell.

[0645] The nucleic acid sequence can be on the same nucleic acid molecule encoding the TFP described herein or be on a different nucleic acid molecule than the one encoding the TFP described herein. For example, the modified T cells can comprise a first nucleic acid sequence encoding a TFP comprising an anti-CD70 antigen binding domain and/or an anti-MSLN antigen binding domain and a second nucleic acid sequence encoding a PD-1 switch molecule. The first nucleic acid sequence and the second nucleic acid sequence can be linked by a linker. In some embodiments, the linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A cleavage site. In some embodiments, the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.

[0646] The PD-1 switch molecule can comprise a PD-1 polypeptide. In some embodiments, the PD-1 polypeptide may be operably linked to the N-terminus of an intracellular domain of a costimulatory polypeptide via the C-terminus of the PD-1 polypeptide. In some embodiments, the costimulatory polypeptide is selected from the group consisting of 0X40, CD2, CD27, CD5, ICAM-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, IL-15Ra, IL12R, IL18R, IL21R, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, and FcyRIII. In some embodiments, the costimulatory peptide is CD28. In some embodiments, an extracellular domain and a transmembrane domain of PD-1 are linked to an intracellular domain of CD28. [0647] In some embodiments, the nucleic acid sequence encodes a PD-1 switch molecule comprising the extracellular domain and the transmembrane domain of PD- 1 linked to the intracellular domain of CD28. In some embodiments, the fusion protein comprises a sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:232 or SEQ ID NO:233. In some embodiments, the fusion protein comprises the sequence of SEQ ID NO:232 or SEQ ID NO:233. In some embodiments, the nucleic acid sequence encodes a PD-1 switch molecule comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15Ra. In some embodiments, the fusion protein comprises a sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:234 or SEQ ID NO:235. In some embodiments, the PD-1 switch molecule comprises the sequence of SEQ ID NO:234 or SEQ ID NO:235.

IL-15 and/or IL-15Ra

[0648] The modified T cells described herein may comprise a nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof. The modified T cells described herein may comprise a nucleic acid sequence encoding an Interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof. In some embodiments, the modified T cells described herein may comprise a nucleic acid sequence encoding a fusion protein comprising an IL- 15 polypeptide or a fragment thereof linked to an IL- 15Ra polypeptide or a fragment thereof. In some embodiments, the modified T cells described herein may comprise a nucleic acid sequence encoding a fusion protein comprising an IL-15Ra polypeptide or a fragment thereof linked to PD-1 or a fragment thereof and/or CD28 or a fragment thereof.

[0649] In some embodiments, the IL- 15 polypeptide or a fragment thereof may comprise an IL- 15 signal peptide. In some embodiments, the IL- 15 polypeptide or a fragment thereof may comprise amino acids 1 -29 of IL- 15. In some embodiments, the IL- 15 polypeptide or a fragment thereof may comprise amino acids 1-29 of SEQ ID NO:256. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:257. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise amino acids 30-162 of IL-15. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise amino acids 30-162 of SEQ ID NO:256. In some embodiments, the IL- 15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:254. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise amino acids 1-162 of SEQ ID NO: 256. In some embodiments, the IL- 15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:257 and a sequence of SEQ ID NO:254. In some embodiments, IL- 15 polypeptide is secreted when expressed in a cell, such as a T cell.

[0650] In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise IL-15Ra signal peptide. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 1-30 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 1-30 of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof does not comprise IL-15Ra signal peptide. In some embodiments, the IL-15Ra polypeptide or a fragment thereof does not comprise amino acids 1-30 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof does not comprise amino acids 1-30 of SEQ ID NO:258.

[0651] In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise IL-15Ra Sushi domain. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 31-95 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 31-95 of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:260.

[0652] In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise an intracellular domain of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 229-267 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 229-267 of a sequence of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:228.

[0653] In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise IL-15Ra Sushi domain, transmembrane domain, and intracellular domain. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 31-267 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 31-267 of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:260. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:261 . In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 96-267 of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:260 and a sequence of SEQ ID NO:261.

[0654] In some embodiments, the IL-15Ra polypeptide or a fragment thereof may be a soluble IL- 15Ra (sIL-15Ra). In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 21-205 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 21-205 of a sequence of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:259.

[0655] The present disclosure encompasses recombinant nucleic acid molecules encoding a fusion protein comprising an IL- 15 polypeptide linked to an IL-15R subunit. In some embodiments, IL- 15 and IL-15R subunit are operatively linked by a linker. In some embodiments, the IL-15R subunit is IL-15R alpha (IL-15Ra). For example, IL-15 polypeptide may be linked to N-terminus of IL-15Ra subunit. For example, IL- 15 polypeptide may be linked to C-terminus of IL-15Ra subunit. In some embodiments, IL-15 and IL-15Ra are operatively linked by a linker. In some embodiments, the linker is not a cleavable linker. For example, the linker may comprise a sequence comprising (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 10. In some embodiments, n is 3. In some embodiments, the linker comprises a sequence of SEQ ID NO:255. In some embodiments, the fusion protein is expressed on cell surface when expressed in a cell, e.g., a T cell. In some embodiments, the fusion protein is secreted when expressed in a cell, e.g., a T cell.

[0656] The nucleic acid sequence can be on the same nucleic acid molecule encoding the TFP described herein or be on a different nucleic acid molecule than the one encoding the TFP described herein. For example, the modified T cells can comprise a first nucleic acid sequence encoding a TFP comprising an anti-CD70 antigen binding domain and/or an anti-MSLN antigen binding domain and a second nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof and/or an IL-15R subunit (e.g., an IL-15 polypeptide linked to an IL-15R subunit, e.g., IL-15Ra). The first nucleic acid sequence and the second nucleic acid sequence can be linked by a linker. In some embodiments, the linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A cleavage site. In some embodiments, the 2A cleavage site is a T2A cleavage site or a P2A cleavage site. Thus, the present disclosure provides a recombinant nucleic acid molecule encoding the first and second TFPs provided herein and/or the first TFP with a first and second antibody domain as provided herein; and further encoding the IL- 15 polypeptide and/or the IL- 15 fusion protein provided herein.

[0657] In some embodiments, the fusion protein may comprise amino acids 30-162 of IL-15. In some embodiments, the fusion protein may comprise amino acids 30-162 of a sequence of SEQ ID NO:256. In some embodiments, the fusion protein may comprise any one of the sequence listed in Table 6 or a fragment thereof. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:254. In some embodiments, the fusion protein does not comprise IL-15 signal peptide. In some embodiments, the fusion protein does not comprise amino acids 1-29 of IL-15. In some embodiments, the fusion protein does not comprise amino acids 1-29 of a sequence of SEQ ID NO:256. In some embodiments, the fusion protein does not comprise a sequence of SEQ ID NO:257.

[0658] In some embodiments, the fusion protein may comprise a Sushi domain. In some embodiments, the fusion protein may comprise amino acids 31-95 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 31-95 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:260.

[0659] In some embodiments, the fusion protein may comprise the intracellular domain of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 229-267 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 229-267 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:228. [0660] In some embodiments, the fusion protein may comprise a soluble IL-15Ra (sIL-15Ra). In some embodiments, the fusion protein may comprise amino acids 21-205 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 21-205 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:259.

[0661] In some embodiments, the fusion protein may comprise the transmembrane domain and the intracellular domain of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 96-267 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 96-267 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:261.

[0662] In some embodiments, the fusion protein may comprise the Sushi domain, the transmembrane domain, and the intracellular domain of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 31-267 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 31-267 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:260 and a sequence of SEQ ID NO:261. In some embodiments, the fusion protein comprising an IL-15 polypeptide or a fragment thereof and an IL-15Ra subunit or a fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NO:263. In some embodiments, the fusion protein comprising an IL- 15 polypeptide or a fragment thereof and an IL-15Ra subunit or a fragment thereof comprises the sequence of SEQ ID NO:263.

Anti-PD-1 Antibodies

[0663] The enhancing agent described herein can be an anti-PD-1 antibody or fragment thereof (e.g., antigen binding fragment). The modified T cells described herein may comprise a nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof that specifically binds programmed cell death protein 1 (PD-1). The anti-PD-1 antibody or fragment thereof can inhibit an interaction of PD-1 with PD-L1 or PD-L2. The anti-PD-1 antibody or fragment thereof can be secreted by the T cell. For example, the modified T cell described herein can comprise a first nucleic acid sequence encoding the TFP described herein and a second nucleic acid sequence encoding the anti-PD-1 antibody or fragment thereof.

[0664] In some embodiments, the anti-PD-1 antibody or fragment thereof comprises a variable domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:238. In some embodiments, the CDR1 comprises the amino acid sequence of SEQ ID NO:236. In some embodiments, the CDR2 comprises the amino acid sequence of SEQ ID NO:237. In some embodiments, the variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:239. In some embodiments, the variable domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:239. In some embodiments, the variable domain comprises the amino acid sequence of SEQ ID NO:239. In some embodiments, the variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 240-243. In some embodiments, the variable domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 240-243. In some embodiments, the variable domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 240-243.

[0665] In some cases, the second nucleic acid encodes a fusion protein comprising an anti-PD-1 antibody or fragment thereof that specifically binds PD-1. For example, the anti-PD-1 antibody or antigen binding fragment thereof may be operably linked to the N-terminus of an intracellular domain of a costimulatory polypeptide via the C-terminus of the anti-PD-1 antibody, or antigen binding fragment thereof. In some embodiments, the anti-PD-1 antibody is linked to the intracellular domain of the costimulatory polypeptide via the transmembrane domain of PD-1.

[0666] In some embodiments, the fusion protein further comprises a signal sequence. In some embodiments, the signal sequence is a PD-1 signal peptide. In some embodiments, the signal sequence comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:244 or SEQ ID NO:245. In some embodiments, the signal sequence comprises the amino acid sequence of SEQ ID NO:244 or SEQ ID NO:245. In some embodiments, the anti-PD-1 antibody or fragment thereof binds to PD-1 on the surface of the T cell, PD-1 on the surface of a bystander T cell, or a combination thereof. In some embodiments, the fusion protein further comprises an intracellular domain operatively linked to the transmembrane domain. In some embodiments, the fusion protein further comprises a transmembrane domain operatively linked to the intracellular domain and the anti-PD-1 antibody or fragment thereof. In some embodiments, the transmembrane domain of the fusion protein comprises a transmembrane domain of a protein selected from the group consisting of CD28, CD3a, CD3^, CD45, CD4, CD5, CD7, CD8, CD9, CD 16, CD22, CD33, CD37, CD41, CD64, CD68, CD80, CD86, CD134, CD137, CD154, ICOS, 4-1BB, 0X40, PD- I, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the transmembrane domain of the fusion protein comprises a PD-1 transmembrane domain. In some embodiments, the transmembrane domain of the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:246. In some embodiments, the transmembrane domain of the fusion protein comprises the amino acid sequence of SEQ ID NO:246. In some embodiments, the fusion protein further comprises a PD-1 stalk domain. In some embodiments, the PD-1 stalk domain is operatively linked to the transmembrane do-main. In some embodiments, the PD-1 stalk domain is operatively linked to the N-terminus of the transmembrane domain. In some embodiments, the PD-1 stalk domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:247. In some embodiments, the PD-1 stalk domain comprises the amino acid sequence of SEQ ID NO:247. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:247 operatively linked to an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:246. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:247 operatively linked to the N terminus of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:246. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:247 operatively linked to the amino acid sequence of SEQ ID NO:246. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:247 operatively linked to the N-terminus of the amino acid sequence of SEQ ID NO:246. In some embodiments, the intracellular domain of the fusion protein comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain comprises a co-stimulatory domain of a protein selected from the group consisting of a CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, PD-1, CD258, ICAM-1, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the co-stimulatory domain comprises a 4-1BB (CD137) co-stimulatory domain. In some embodiments, the co-stimulatory domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:224. In some embodiments, the co- stimulatory domain comprises the amino acid sequence of SEQ ID NO:224. In some embodiments, the co-stimulatory domain comprises a CD28 co-stimulatory domain. In some embodiments, the co- stimulatory domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:223. In some embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:223. In some embodiments, the fusion protein comprises two or more anti -PD-1 antibodies or fragments thereof. In some embodiments, the two or more anti -PD-1 antibodies or fragments thereof are operatively linked tandemly. In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are identical. In some embodiments, the two or more anti-PD- 1 antibodies or fragments thereof are different. In some embodiments, the two or more anti- PD-1 antibodies or fragments thereof are operatively linked by a linker. In some embodiments, the linker comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:248. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:248. In some embodiments, the fusion protein further comprises a PD-1 signal peptide, a PD-1 stalk domain, a PD-1 transmembrane domain, and a CD28 or 4-1BB (CD137) co-stimulatory do-main. In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a PD-1 signal peptide operatively linked to the anti-PD-1 antibody or fragment thereof operatively linked to a PD-1 stalk domain operatively linked to a PD-1 transmembrane domain operatively linked to a CD28 or 4-1BB (CD137) co-stimulatory domain. In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a PD-1 signal peptide operatively linked to a first anti-PD- 1 antibody or fragment thereof operatively linked to a linker operatively linked to a second anti-PD-1 antibody or fragment thereof operatively linked to a PD-1 stalk domain operatively linked to a PD-1 transmembrane domain operatively linked to a CD28 or 4-1BB (CD 137) co-stimulatory domain. In some embodiments, the fusion protein comprises SEQ ID NO:244 or SEQ ID NO:245, SEQ ID NO:239 or SEQ ID NO:241, SEQ ID NO:247, SEQ ID NO:246, and SEQ ID NO:223 or SEQ ID NO:224. In some embodiments, the fusion protein comprises, from the N-terminus to the C- terminus, SEQ ID NO:244 or SEQ ID NO:245 operatively linked to SEQ ID NO:239 or SEQ ID NO:241 operatively linked to SEQ ID NO:247 operatively linked to SEQ ID NO:246 operatively linked to SEQ ID NO:223 or SEQ ID NO:224. In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, SEQ ID NO:244 or SEQ ID NO:245 operatively linked to SEQ ID NO:239 or SEQ ID NO:241 operatively linked to SEQ ID NO:248 operatively linked to SEQ ID NO:239 or SEQ ID NO:241 operatively linked to SEQ ID NO:247 operatively linked to SEQ ID NO:246 operatively linked to SEQ ID NO:223 or SEQ ID NO:224. In some embodiments, the fusion protein comprises an amino acid sequence having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to the amino acid sequence of any one of SEQ ID NOs:249-253. In some embodiments, the fusion protein comprises an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs:249-253.

Sources of T cells

[0667] 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 (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells can be obtained from a leukopak. In certain aspects of the present disclosure, any number of T cell lines available in the art, may be used. In certain aspects of the present disclosure, 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 present disclosure, 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 OncologyCytoMate, 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 media.

[0668] In embodiments, the T cells are <x|3 T cells. In some embodiments, the T cells are y5 T cells. y5 T cells are obtained from a bank of umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cells, for example.

[0669] 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+, CD45RO+, alpha-beta, or gamma-delta T cells, can be further isolated by positive or negative selection techniques. For example, in one aspect, CD4+ and CD8+ T cells are isolated with anti-CD4 and anti-CD8 microbeads. In another 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 or Trans-Act® beads, 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 present disclosure. 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.

[0670] 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. [0671] In one embodiment, a T cell population can be selected that expresses one or more of IFN-y TNF-alpha, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-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. WO2013126712, which is herein incorporated by reference.

[0672] 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/mL is used. In one aspect, a concentration of 1 billion cells/mL is used. In a further aspect, greater than 100 million cells/mL is used. In a further aspect, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In yet one aspect, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mL is used. In further aspects, concentrations of 125 or 150 million cells/mL 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.

[0673] 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 are 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 5xlO ⁶ /mL. In other aspects, the concentration used can be from about IxlO mL to lxlO 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.

[0674] 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 disclosure.

[0675] Also contemplated in the context of the present disclosure 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 CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporin, rapamycin, mycophenolic acid, steroids, romidepsin (formerly FR901228), and irradiation.

[0676] In a further aspect of the present disclosure, 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 fortheir 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 disclosure 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

[0677] 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.

[0678] Generally, the T cells of the present disclosure may be expanded by contact with a surface having atached 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 antigenbinding 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 costimulation 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, CD8+ T cells, or CD4+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 al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol. Meth. 227( 1-2): 53-63, 1999). T cells may additionally be activated and expanded in the presence of a cytokine with or without an anti-CD3 and/or CD28 antibody. Exemplary cytokines include IL-2, IL-7, IL-15, and IL-21. In some embodiments, T cells are activated by incubation with anti-CD3/anti-CD28-conjugated beads, such as DYNABEADS® or Trans-Act® beads, for a time period sufficient for activation of the T cells. In one 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, e.g., 24 hours. In some embodiments, T cells are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with cytokines that bind the common gammachain (e.g., IL-2, IL-7, IL-12, IL-15, IL-21, and others). In some embodiments, T cells are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 100 U/mL of IL-2, IL-7, and/or IL-15. In some embodiments, the cells are activated for 24 hours. In some embodiments, after transduction, the cells are expanded in the presence of anti-CD3 antibody, anti-CD28 antibody in combination with the same cytokines. In some embodiments, cells activated in the presence of an anti- CD3 antibody and an anti-CD28 antibody in combination with cytokines that bind the common gamma-chain are expanded in the presence of the same cytokines in the absence of the anti-CD3 antibody and anti-CD28 antibody after transduction. In some embodiments, after transduction, the cells are expanded in the presence of anti-CD3 antibody, anti-CD28 antibody in combination with the same cytokines up to a first washing step, when the cells are sub-cultured in media that includes the cytokines but does not include the anti-CD3 antibody and anti-CD28 antibody. In some embodiments, the cells are subcultured every 1, 2, 3, 4, 5, or 6 days. In some embodiments, cells are expanded for 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, or 30 days. [0679] The expansion of T cells may be stimulated with zoledronic acid (Zometa), alendronic acid (Fosamax) or other related bisphosphonate drugs at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10, or 100 pM in the presence of feeder cells (irradiated cancer cells, PBMCs, artificial antigen presenting cells). The expansion of T cells may be stimulated with isopentyl pyrophosphate (IPP), (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP or HMB-PP) or other structurally related compounds at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10, or 100 pM in the presence of feeder cells (irradiated cancer cells, PBMCs, artificial antigen presenting cells). In some embodiments, the expansion of T cells may be stimulated with synthetic phosphoantigens (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1 PP, or 2-methyl-3-butenyl-l -pyrophosphate in the presence of IL-2 for one-to-two weeks. In some embodiments, the expansion of T cells may be stimulated with immobilized anti-TCRyd (e.g., pan TCRY6) in the presence of IL-2, e.g., for approximately 14 days. In some embodiments, the expansion of T cells may be stimulated with culture of immobilized anti-CD3 antibodies (e.g., OKT3) in the presence of IL-2. In some embodiments, the aforementioned culture is maintained for about seven days prior to subculture in soluble anti-CD3, and IL-2.

[0680] T cells that have been exposed to varied stimulation times may exhibit different characteristics. Lor 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.

[0681] Eurther, 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.

[0682] 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.

[0683] 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)). Very briefly, 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.

[0684] 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 as described herein along with eGFP using a 2A ribosomal skipping sequence. In some embodiments, Cultures are re-stimulated with either TAA+ K562 cells (K562-TAA), wild-type K562 cells (K562 wild type) or K562 cells expressing hCD32 and 4-1BBL in the presence of anti-CD3 and anti-CD28 antibody (K562 -BBL-3/28) following washing. Exogenous IL-2 is added to the cultures every other day at 100 lU/mL. GFP+ T cells are enumerated by flow cytometry using bead-based counting (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).

[0685] 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 as described herein on day 1.

[0686] Animal models can also be used to measure an activity of T cells expressing TFT as described herein. For example, xenograft model using, e.g., human CD70 and/or MSLN-specific TFP+ T cells to treat a cancer in immunodeficient mice (see, e.g., 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 anti -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 as described herein by a bicistronic lentiviral vector that encodes the TFP linked to eGFP. T cells are normalized 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 1-week intervals. Survival curves for the TFP+ T cell groups are compared using the log-rank test.

[0687] Dose dependent TFP treatment response can be evaluated (see, e.g., Milone et al., 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. [0688] Assessment of cell proliferation and cytokine production has been previously described, e.g., at Milone et al., 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 the tumor associated antigen (TAA, e.g., CD70 and/or MSLN) for a final T cell: cell expressing TAA ratio of 2: 1. Cells expressing TAA 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 CountB right™ 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 TAA 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 (BD Biosciences), and data are analyzed according to the manufacturer’s instructions.

[0689] Cytotoxicity can be assessed by a standard ⁵¹ Cr-release assay (see, e.g. , Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Briefly, target cells are loaded with ⁵¹ Cr (as NaCrCft, 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.

[0690] 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 al., Human Gene Therapy 22: 1575-1586 (2011). Briefly, NOD/SCID/yc-/- (NSG) mice are injected IV with cancer cells followed 7 days later with T cells 4 hour after electroporation with the TFP as described herein 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 tailvein injection of T cells electroporated with a TAA-TFP as described herein 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.

[0691] 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 of the present disclosure.

Therapeutic Applications

[0692] The TFP T cells provided herein may be useful for the treatment of any disease or condition involving CD70 and/or MSLN (e.g., Nectin-4-expressing cancers). In some embodiments, the disease or condition is a disease or condition that can benefit from treatment with adoptive cell therapy. In some embodiments, the disease or condition is a cell proliferative disorder. In some embodiments, the disease or condition is a cancer. In some embodiments, the disease or condition is a blood cancer. In some embodiments, the disease or condition is a tumor. In some embodiments, the disease or condition is a viral infection.

[0693] In some embodiments, provided herein is a method of treating a disease or condition in a subject in need thereof by administering an effective amount of a TFP T cell provided herein to the subject. In some aspects, the disease or condition is a cancer. In some aspects, the disease or condition is a viral infection.

[0694] Many patients treated with cancer therapeutics that are directed to one target on a tumor cell, e.g., BCMA, CD70, CD19, CD20, CD22, CD123, MUC16, MSLN, 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 some embodiments, the dual specificity TFP T cells are administered with an additional anti-cancer agent; in some embodiments, the anticancer agent is an antibody or fragment thereof, another TFP T cell, a CAR T cell, or a small molecule. 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), posttranslationally 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, ARTCI, alphafetoprotein (AFP), BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDK12, CDKN2A, CLPP, COA-1, CSNK1A1, CD79, CD79B, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-

- I l l - AML1 fusion protein, FLT3-ITD, FNDC3B, FN1, GAS7, GPNMB, HAUS3, HSDL1, LDLR- fucosyltransferase AS fusion protein, HLA-A2d, HLA-A1 Id, hsp70-2, MART2, MATN, MEI, 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-1, D393-CD20n, Cyclin-Al, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GnTVf, HERV-K- MEL, KK-LC-1, KM-HN-1, LAGE-1, LY6K, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12 m, MAGE-CI, MAGE-C2, mucink, NA88-A, NY-ESO-1 / LAGE-2, SAGE, Spl7, SSX-2, SSX-4, TAG-1, TAG-2, TRAG-3, TRP2-INT2g, XAGE-lb/GAGED2a, , Gene / protein, CEA, gplOO / Pmell7, mammaglobin-A, Melan-A / MART- 1, NY-BR-1, OA1, PAP, PSA, RAB38 / NY-MEL-1, TRP-1 / gp75, TRP-2, tyrosinase, adipophilin, AIM-2, ALDH1A1, BCLX (L), BING-4, CALCA, CD45, CD274, CPSF, cyclin DI, DKK1, ENAH (hMena), EpCAM, EphA3, EZH2, FGF5, glypican-3, G250 / MN / CAIX, HER-2 / neu, HLA-DOB, Hepsin, IDO1, IGF2B3, IL13Ralpha2, 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-1, RGS5, RhoC, RNF43, RU2AS, secemin 1, SOXIO, STEAP1, survivin, Telomerase, TPBG, VEGF, and WT1.

[0695] In one aspect, the present disclosure provides methods for treating a disease associated with at least one tumor-associated antigen expression. In one aspect, the present disclosure 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 present disclosure 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.

[0696] In some embodiments, provided herein is a method of treating a disease or condition in a subject in need thereof by administering an effective amount of a TFP T cell provided herein to the subject. In some aspects, the disease or condition is a cancer. In some aspects, the disease or condition is a viral infection.

[0697] Any suitable cancer may be treated with the TFP T cells provided herein. Illustrative suitable cancers include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fimgoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor.

[0698] In some cases, the cancer can be acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia (CLL), chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastric cancer, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), glioblastoma, Hodgkin's lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung carcinoma), lymphoma, diffuse large-B-cell lymphoma, follicular lymphoma, malignant mesothelioma, mastocytoma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin's lymphoma (NHL), B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, mesentery cancer, pharynx cancer, prostate cancer, RCC, ccRCC, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, or ureter cancer. The cancer can be characterized by the expression CD70 and/or MSLN. In some cases, CD70 and/or -MSLN can be highly expressed in breast cancer, lung cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, esophageal cancer, head and neck cancer, bladder cancer, and gastric cancer.

[0699] In some embodiments, the disease or the condition is a cancer or a disease or a condition associated with expression of mesothelin (MSLN) and/or CD70. In some embodiments, the cancer is a hematologic cancer. Examples of a hematologic cancer include, but are not limited to, B-cell acute lymphoid leukemia (B-ALL), T cell acute lymphoid leukemia (T-ALL), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell pro- lymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell-follicular lymphoma, large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodys-plastic syndrome, non-Hodgkin’s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and preleukemia. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0700] In one aspect, the present disclosure 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 present disclosure 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. In one aspect, the tumor-associated antigen TFP-T 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 targets the tumor cell and growth of the tumor is inhibited.

[0701] In one aspect, the present disclosure 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.

[0702] 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.

[0703] 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.

[0704] 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.

[0705] 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.

[0706] 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.

[0707] 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.

[0708] 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.

[0709] 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.

[0710] The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described, e.g., in U.S. Pat. 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. Pat. No. 5,199,942, other factors such as flt3-U, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.

[0711] 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.

[0712] 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. [0713] In one aspect, the antibodies or TFP-T cells of the present disclosures 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.

[0714] 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 or IL- 12 or other cytokines or cell populations.

[0715] 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.

[0716] 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).

[0717] 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.

[0718] 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 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.

[0719] Suitable doses of the TFP-T cells described herein for a therapeutic effect would be at least 10 ⁵ or between about 10 ⁵ and about 10 ¹⁰ cells per dose, for example, preferably in a series of dosing cycles. An exemplary dosing regimen consists of four one-week dosing cycles of escalating doses, starting at least at about 10 ⁵ cells on Day 0, for example increasing incrementally up to a target dose of about 10 ¹⁰ cells within several weeks of initiating an intra-patient dose escalation scheme. Suitable modes of administration include intravenous, subcutaneous, intracavitary (for example by reservoiraccess device), intraperitoneal, and direct injection into a tumor mass.

[0720] An effective amount or sufficient number of the isolated, T cells is present in the composition and introduced into the subject such that long-term, specific, anti -cancer and/or anti -tumor responses are established to reduce the size of a tumor or eliminate tumor growth or regrowth than would otherwise result in the absence of such treatment. Desirably, the amount of T cells introduced into the subject causes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in tumor size when compared to otherwise same conditions wherein the T cells are not present.

[0721] Accordingly, the amount of T cells administered should take into account the route of administration and should be such that a sufficient number of the T cells will be introduced so as to achieve the desired therapeutic response. Furthermore, the amounts of each active agent included in the compositions described herein (e.g., the amount per each cell to be contacted or the amount per certain body weight) can vary in different applications.

Combination Therapies

[0722] An antibody or 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.

[0723] 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 expresses a second TFP.

[0724] 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.

[0725] 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, and tacrolimus, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cyclophosphamide, fludarabine, cyclosporin, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, cytokines, and irradiation, peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg 108:963-971. [0726] 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-y, TNFa, IL-2 and IL-6. 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®).

[0727] 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 PD 1 , PD-L 1 , 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).

[0728] 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 antitumor-associated antigen TFP.

[0729] In some embodiments, the additional therapeutic agent comprises an immunostimulatory agent.

[0730] In some embodiments, the immunostimulatory agent is an agent that blocks signaling of an inhibitory receptor of an immune cell, or a ligand thereof. In some aspects, the inhibitory receptor or ligand is selected from cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), programmed cell death protein 1 (also PD-1 or CD279), programmed death ligand 1 (also PD-L1 or CD274), transforming growth factor beta (TGF[3), lymphocyte-activation gene 3 (LAG-3, also CD223), Tim-3 (hepatitis A virus cellular receptor 2 or HAVCR2 or CD366), neuritin, B- and T- lymphocyte attenuator (also BTLA or CD272), killer cell immunoglobulin-like receptors (KIRs), and combinations thereof. In some aspects, the agent is selected from an anti -PD-1 antibody (e.g., pembrolizumab or nivolumab), and anti-PD-Ll antibody (e.g., atezolizumab), an anti-CTLA-4 antibody (e.g., ipilimumab), an anti-TIM3 antibody, carcinoembryonic antigen-related cell adhesion molecule 1 (CECAM-1, also CD66a) and 5 (CEACAM-5, also CD66e), vset immunoregulatory receptor (also VISR or VISTA), leukocyte-associated immunoglobulin-like receptor 1 (also LAIR1 or CD305), CD 160, natural killer cell receptor 2B4 (also CD244 or SLAMF4), and combinations thereof. In some aspects, the agent is pembrolizumab. In some aspects, the agent is nivolumab. In some aspects, the agent is atezolizumab.

[0731] In some embodiments, the additional therapeutic agent is an agent that inhibits the interaction between PD-1 and PD-L1. In some aspects, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is selected from an antibody, a peptidomimetic and a small molecule. In some aspects, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is selected from pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), atezolizumab, avelumab, pidilizumab, durvalumab, sulfamonomethoxine 1, and sulfamethizole 2. In some embodiments, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is any therapeutic known in the art to have such activity, for example as described in Weinmann et al., ChemMed Chem, 2016, 14: 1576 (DOI: 10.1002/cmdc.201500566), incorporated by reference in its entirety. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in the same pharmaceutical composition an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in a different pharmaceutical composition from an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is administered prior to administration of an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is administered after administration of an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is administered contemporaneously with an antibody provided herein, but the agent and antibody are administered in separate pharmaceutical compositions.

[0732] In some embodiments, the immunostimulatory agent is an agonist of a co-stimulatory receptor of an immune cell. In some aspects, the co-stimulatory receptor is selected from GITR, 0X40, ICOS, LAG-2, CD27, CD28, 4-1BB, CD40, STING, a toll-like receptor, RIG-1, and a NOD-like receptor. In some embodiments, the agonist is an antibody.

[0733] In some embodiments, the immunostimulatory agent modulates the activity of arginase, indoleamine-2 3 -dioxygenase, or the adenosine A2A receptor.

[0734] In some embodiments, the immunostimulatory agent is a cytokine. In some aspects, the cytokine is selected from IL-2, IL-5, IL-7, IL- 12, IL- 15, IL-21, and combinations thereof.

[0735] In some embodiments, the immunostimulatory agent is an oncolytic virus. In some aspects, the oncolytic virus is selected from a herpes simplex virus, a vesicular stomatitis virus, an adenovirus, a Newcastle disease virus, a vaccinia virus, and a maraba virus.

[0736] Further examples of additional therapeutic agents include a taxane (e.g., paclitaxel or docetaxel); a platinum agent (e.g., carboplatin, oxaliplatin, and/or cisplatin); a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, and/or mitoxantrone); folinic acid (e.g., leucovorin); or a nucleoside metabolic inhibitor (e.g., fluorouracil, capecitabine, and/or gemcitabine). In some embodiments, the additional therapeutic agent is folinic acid, 5 -fluorouracil, and/or oxaliplatin. In some embodiments, the additional therapeutic agent is 5 -fluorouracil and irinotecan. In some embodiments, the additional therapeutic agent is a taxane and a platinum agent. In some embodiments, the additional therapeutic agent is paclitaxel and carboplatin. In some embodiments, the additional therapeutic agent is pemetrexate. In some embodiments, the additional therapeutic agent is a targeted therapeutic such as an EGFR, RAF or MEK-targeted agent.

[0737] The additional therapeutic agent may be administered by any suitable means. In some embodiments, a medicament provided herein, and the additional therapeutic agent are included in the same pharmaceutical composition. In some embodiments, an antibody provided herein, and the additional therapeutic agent are included in different pharmaceutical compositions.

[0738] In embodiments where an antibody provided herein and the additional therapeutic agent are included in different pharmaceutical compositions, administration of the antibody can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about one month of each other. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about one week of each other. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about one day of each other. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about twelve hours of each other. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about one hour of each other.

[0739] In some embodiments, the agent is an agent that inhibits DNA methylation. In some embodiments, the agent is an agent that inhibits DNA methyltransferease. In some embodiments, the agent is a hypomethylating agent. Examples of the hypomethylating agent includes, but are not limited to 5 -azacitidine and decitabine and also includes any hypomethylating agent known in the art. In some embodiments, the hypomethylating agent is 5 -azacitidine. In some embodiments, the hypomethylating agent is decitabine. In some embodiments, the hypomethylating agent is a derivative of decitabine or a derivative of 5 -azacitidine. In some embodiments, the hypomethylating agent is an esterificated azacytidine, an acetylated azacitidine, an esterificated decitabine. or an acetylated decitabine.

Pharmaceutical Compositions

[0740] 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.

[0741] 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.

[0742] 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.

[0743] When “an immunologically effective amount,” “an anti-tumor effective amount,” “a tumorinhibiting 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 IO ⁴ to IO ⁹ 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 al., New Eng. J. of Med. 319: 1676, 1988).

[0744] 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.

[0745] 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.

[0746] 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.

[0747] 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. Pat. No. 6,120,766).

[0748] In one embodiment, the TFP as described herein 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., 1, 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.

[0749] 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. [0750] 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.

[0751] 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. [0752] 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.

[0753] 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

[0754] 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. The following Examples describe engineered T cell receptors having specificity for more than one target antigen on a cancer cell; in addition are described methods of creating populations of T cells having TCRs specific for more than one antigen, either in the same cell or in a combination of cells. In one embodiment, TFP constructs are made having both binding domains (e.g., an scFv, a sdAb, etc.) in tandem on a single TCR subunit. In one embodiment, TFP constructs are made having both binding domains in a single TCR with one binding domain on each of two TCR subunits, e.g., both epsilon subunits, an epsilon and the gamma subunit, etc. In another embodiment, TFP constructs are made individually in separate lentiviral vectors, and the target T cell population is transduced with both viruses. The Examples disclose a combination of anti-MSLN TFPs and anti-CD70 TFPs and/or a TFP having specificity to both anti-MSLN and CD70, and/or a mixed T cell population wherein the T cells are a mix of T cells transduced with an anti-MSLN TFP and T cells transduced with an anti-CD70 TFP. The anti-MSLN and anti-CD70 constructs disclosed herein are exemplary only and not meant to be construed as limiting, as noted above. Constructs with a variety of combinations of anti-tumor antigen antibodies are contemplated in the methods of the present disclosure.

Source of TCR Subunits

[0755] Subunits of the human T Cell Receptor (TCR) complex all contain an extracellular domain and a transmembrane domain. The CD3 epsilon, CD3 delta, and CD3 gamma subunits have an intracellular domain. A human TCR complex contains the CD3-epsilon polypeptide, the CD3-gamma poly peptide, the CD3-delta polypeptide, and the TCR alpha chain polypeptide and the TCR beta chain polypeptide or the TCR delta chain polypeptide and the TCR gamma chain polypeptide. TCR alpha, TCR beta, TCR gamma, and TCR delta recruit the CD3 zeta 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. Q6ISU 1. The human TCR beta chain C region canonical sequence is Uniprot Accession No. P01850, a human TCR beta chain V region sequence is P04435.

[0756] The human CD3 -epsilon polypeptide canonical sequence is: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQ H NDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCEN CM EMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPV P NPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:694).

[0757] The mature human CD3 -epsilon polypeptide sequence is: DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHL SLK EFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLL LL VYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:733).

[0758] The signal peptide of human CD3a is:

MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO:695).

[0759] The extracellular domain of human CD3a is:

DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDE DHLSLK

EFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO:696).

[0760] The transmembrane domain of human CD3a is:

VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID NO: 697).

[0761] The intracellular domain of human CD3s is:

KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:698).

[0762] The human CD3 -gamma polypeptide canonical sequence is:

MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFK DGKMI GFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAE I VSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO:699).

[0763] The mature human CD3 -gamma polypeptide sequence is:

QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAK DPR GMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGV RQ SRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO:734).

[0764] The signal peptide of human CD3y is:

MEQGKGLAVLILAIILLQGTLA (SEQ ID NO: 700).

[0765] The extracellular domain of human CD3y is:

QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAK DPR

GMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS (SEQ ID NO:701).

[0766] The transmembrane domain of human CD3y is:

GFLFAEIVSIFVLAVGVYFIA (SEQ ID NO:702).

[0767] The intracellular domain of human CD3y is:

GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO:703).

[0768] The human CD3 -delta polypeptide canonical sequence is:

MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITR LDLGKRIL DPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCF AGH ETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKS (SEQ ID NO:704).

[0769] The mature human CD3 -delta polypeptide sequence is:

FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDI YKDKESTV

QVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALL RNDQV YQPLRDRDDAQYSHLGGNWARNKS (SEQ ID NO:735).

[0770] The signal peptide of human CD35 is: MEHSTFLSGLVLATLLSQVSP (SEQ ID NO:705).

[0771] The extracellular domain of human CD35 is:

FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDI YKDKESTV

QVHYRMCQSCVELDPATVA (SEQ ID NO:706).

[0772] The transmembrane domain of human CD35 is:

GIIVTDVIATLLLALGVFCFA (SEQ ID NO:707).

[0773] The intracellular domain of human CD35 is:

GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO:708).

[0774] The human CD3-zeta polypeptide canonical sequence is:

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSR SADAPAY

QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAE AY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:709).

[0775] The human TCR alpha chain canonical sequence is:

MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGL DSPI

WFSAGNGSALDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSR STQP MHLSGEASTARTCPQEPLRGTPGGALWLGVLRLLLFKLLLFDLLLTCSCLCDPAGPLPSP ATT TRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVWGEGSYLSSYPT CP

AQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGAA (SEQ ID NO:710).

[0776] The human TCR alpha chain C region canonical sequence is:

IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFK SNSAV

AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF RILLLKVA GFNLLMTLRLWSS (SEQ ID NO: 711).

[0777] The human TCR alpha chain human IgC sequence is:

IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFK SNSAV

AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS (SEQ ID NO:712).

[0778] The transmembrane domain of the human TCR alpha chain is:

VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO:713).

[0779] The intracellular domain of the human TCR alpha chain is:

SS

[0780] The human TCR alpha chain V region CTL-L17 canonical sequence is:

MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFD YFLW

YKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAA KGAGTAS KLTFGTGTRLQVTL (SEQ ID NO:714).

[0781] The human TCR beta chain C region canonical sequence is:

EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVS TDPQP LKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS A EAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO:715).

[0782] The human TCR beta chain constant region is:

VEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGV STDPQ PLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIV SA EAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO:211).

[0783] The human TCR beta chain human IgC sequence is:

EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVS TDPQP LKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS A EAWGRADCGFTSVSYQQGVLSATILYE (SEQ ID NO: 716).

[0784] The transmembrane domain of the human TCR beta chain is: ILLGKATLYAVLVSALVLMAM (SEQ ID NO: 717).

[0785] The human TCR beta chain V region CTL-L17 canonical sequence is:

MGTSLLCWMALCLLGADHADTGVSQNPRHNITKRGQNVTFRCDPISEHNRLYWYRQT LGQ GPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLAGLNQ PQH FGDGTRLSIL (SEQ ID NO: 718).

[0786] The intracellular domain of the human TCR beta chain is:

VKRKDF (SEQ ID NO:719).

[0787] The human TCR beta chain V region YT35 canonical sequence is:

MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQT MMRG LELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSFSTCSAN YGYT FGSGTRLTVV (SEQ ID NO: 720).

[0788] The human TCR gamma chain C region canonical sequence is:

DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGS QEGNTMK TNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKD AN DTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS (SEQ ID NO:721).

[0789] The human TCR gamma human IgC sequence is:

DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGS QEGNTMK TNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKD AN DTLLLQLTNTSA (SEQ ID NO: 722).

[0790] The transmembrane domain of the human TCR gamma chain is: YYMYLLLLLKSVVYFAIITCCLL (SEQ ID NO:723).

[0791] The intracellular domain of the human TCR gamma chain is:

RRTAFCCNGEKS (SEQ ID NO:724).

[0792] The human TCR delta chain C region canonical sequence is:

SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNA VKLGKYE DSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNM M SLTVLGLRMLFAKTVAVNFLLTAKLFFL (SEQ ID NO:725). [0793] The human TCR delta human IgC sequence is:

SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNA VKLGKYE DSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNM M SLTV (SEQ ID NO:726).

[0794] The transmembrane domain of the human TCR delta chain is: LGLRMLFAKTVAVNFLLTAKLFF (SEQ ID NO:727).

[0795] The intracellular domain of the human TCR delta chain is:

L

[0796] The murine TCR alpha chain constant (mTRAC) region canonical sequence is:

XIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDS KSNGAI AWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKV AG FNLLMTLRLWSS (SEQ ID NO:212).

[0797] The murine TCR alpha chain (2-137) sequence is:

IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSK SNGAIA WSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVA GF NLLMTLRLWSS (SEQ ID NO:213).

[0798] The murine TCR beta chain constant region canonical sequence (murine TCR beta chain constant region 1 canonical sequence) is:

EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVS TDPQ AYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEA W GRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO:214).

[0799] The murine TCR beta chain (2-173) sequence is:

DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVST DPQA YKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAW G RADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO:215). [0800] The transmembrane domain of the murine TCR alpha chain is:

MGLRILLLKVAGFNLLMTLRLW (SEQ ID NO:736).

[0801] The transmembrane domain of the murine TCR beta chain 1 is: ILYEILLGKATLYAVLVS TLVVMAMVK (SEQ ID NO:738).

[0802] The intracellular domain of the murine TCR beta chain 1 is:

KRKNS (SEQ ID NO:739).

[0803] The murine TCR beta chain constant 2 region canonical sequence is:

XDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVS TDPQ AYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEA W GRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO:740). Example 1: Design of CD70 and MSLN targeting bispecific T cell receptor (TCR) fusion constructs (TRuC ™)

[0804] In the solid tumor microenvironment, single antigen targeting cell therapies (e.g., T cell therapy) encounter a heterogeneous milieu, which can challenge the efficacy or persistence of the treatment. To test whether a bispecific T cell receptor (TCR) fusion construct (TRuC) can mitigate antigen escape and improve antitumor activity, TRuCs specifically targeting both CD70 and mesothelin (MSLN) were designed and tested. The general methods used have previously been described in International Publications WO2018067993, WO2018098365, W02020047501, and International Application PCT/US21/30973, the contents of each of which are incorporated herein by reference in their entirety.

[0805] A set of 16 initial constructs (SEQ ID NOs: 1001-1016) was designed to test various configurations and optimizations for bispecific TRuCs targeting CD70 and MSLN and these and the components thereof are outlined in Table 2. The CD70 binder selected for use was the CIO scFv comprising a light chain variable domain sequence given as SEQ ID NO:368, encoded by any one of SEQ ID NOs: 1042, 1061, and 1073, and a heavy chain variable domain sequence given as SEQ ID NO:364, encoded by any one of SEQ ID NOs: 1044, 1063, and 1075. A CD70 targeting mono TRuC was used as a control (SEQ ID NO: 1002). The primary MSLN binder selected for use was the MH1 variable heavy homodimer (VHH) given as SEQ ID NO:69, encoded by any one of SEQ ID NO: 1039, 1057, 1069, and 1081. Alternative MSLN binder MH4 (SEQ ID NO:70), encoded by SEQ ID NO: 1059 was also used as a reference. A MSLN targeting mono TRuC was used as a control (SEQ ID NO: 1001). Together, the constructs were designed to test function, format, orientation, composition, and codon-optimization. To test format, dual TRuC (two individual TRuCs each with a single antigen binder operatively linked to a TCR subunit) and tandem binder designs (two antigen binders tandemly linked to a single TCR subunit) were prepared. The effects of orientation were assessed by altering the configuration when read 5’ to 3’ or N- terminus to C- terminus, in respect to binders and/or TCR subunit sequences. Linker sequences and cleavage sites were exchanged (e.g., T2A versus P2A) and inclusion of a furin cleavage site was tested in two constructs. Codon-optimized variants were also generated (“Optil” and “Opti2”). Schematics of various embodiments of the CD70/MSLN dual TRuC designs are shown in Figure 1. Sequences of the exemplary CD70/MSLN dual TRuC designs are provided in Table 2.

[0806] Each construct was cloned by standard methods into a pLRPO, pLKaUS, or pLCUS lentiviral vector additionally comprising a ubiquitous promoter derived from cytomegalovirus (CMV), an intron derived from the elongation factor 1 alpha gene, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and transduced into CD3/CD28 activated T-cells for generation of the associated TRuC T cells.

Example 2: Dual TRuC expression and function in vitro

[0807] To test expression and function of an exemplary CD70/MSLN targeting bispecific TRuC in vitro, a C10s-2A-MHly construct (SEQ ID NO: 1003) was selected and compared to mono TRuCs MHly (SEQ ID NO: 1001), CIOs (SEQ ID NO: 1002) and MHls (SEQ ID NO: 195) (having an anti- MSLN VHH binder fused to CD3e).

[0808] First, a standard T cell expansion assay was conducted, wherein T cells collected from healthy control samples were transduced with each of the constructs (or a non-transduced control) and allowed to expand in the presence of IL-7 and IL- 15 for ten days. Fold expansion was determined over time and was substantially similar across all conditions. These findings suggested that T cell expansion was not substantially impeded by the expression of a mono TRuC orthe C10s-2A-MHly dual TRuC.

[0809] T cells were then collected for measurement by flow cytometry to determine the expression of the C10s-2A-MHly dual or mono TRuCs. The data are shown in Figure 2 and demonstrate that MSLN binder MH1 and CD70 binder CIO were each successfully detected subsequent to transduction with mono TRuCs MHls, MHly, or CIOs, respectively. Transduction with the C10s-2A-MHly dual TRuC yielded detection of both CIO and MH1 by flow cytometry. These data were indicative of successful expression of mono or dual TRuC constructs in T cells.

[0810] Cytotoxicity of C10s-2A-MHly dual TRuC or mono TRuC T cells was evaluated by 24hr coculture with CD70 and/or MSLN expressing cell lines and inverse quantification of cell lysis based on a luciferase readout. The 786-0 cell line was selected for high CD70 expression (CD70 ^Hlgh ), while MSTO-MSLN was selected for high MSLN expression (MSLN ^Hlgh ). A 1: 1 mixture of 786-0 and MSTO-MSLN cells was used as the test condition for dual targeting. TRuC T cells and target cells were co-cultured at 3: 1, 1: 1 or 1:3 ratios, respectively and the calculated percent cytotoxicity is shown in Figure 3. In the co-culture with 786-0 cells (CD70 ^Hlgh ), the CIOs mono TRuC and the C10s-2A- MHly dual TRuC showed the highest % cytotoxicity. Meanwhile, in the co-culture with MSTO- MSLN (MSLN ^Hlgh ) cells, MHls, MHly, and C10s-2A-MHly showed the greatest efficacy. In coculture of 786-0, MSTO-MLSN and TRuC T cells, the CIOs mono TRuC and the C10s-2A-MHly dual TRuC again showed the highest % cytotoxicity. These data showed effective targeting of each of the CD70 and MSLN antigens by the C10s-2A-MHly dual TRuC, much like the targeting evident with mono TRuCs directed toward either CD70 or MSLN alone.

[0811] Following the cytotoxicity assay, the supernatant from each condition was collected and assessed for cytokine release as determined by quantification of interferon gamma (IFNy), interleukin- 2 (IL-2), tumor necrosis factor alpha (TNFa) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Co-culture of 786-0 cells (CD70 ^Hlgh ) with CIOs mono TRuC T cells showed the highest release of IFNy, IL-2, TNFa, and GM-CSF, while co-culture with CI0s-2A-MHIy dual TRuC T cells showed slightly less. Cytokine release in each of these conditions scaled to the ratio of TRuC T cells to target cells, with the greatest response at the 3: 1 ratio and the least at the 1:3 ratio. Co-culture of 786-0 with MHls or MHly mono TRuC T cells generated negligible cytokine release when compared to the other conditions. Similarly, co-culture of the MSTO-MSLN cells (MSLN ^Hlgh ) with MHly mono TRuC T cells generated the greatest cytokine release for each of IFNy, IL-2, TNFa, and GM-CSF cytokines. Co-culture of MSTO-MSLN with either MHls or C10s-2A-MHly dual TRuC T cells showed less of an effect on cytokine release. Co-culture of MSTO-MSLN with CIOs mono TRuC T cells gave negligible cytokine release when compared to the other conditions. In the coculture of 786-0, MSTO-MSLN and mono or dual TRuC T cells, the CIOs mono TRuC and the C10s-2A-MHly dual TRuC T cells generally showed the greatest induction of cytokine release, as measured by IFNy, IL-2, TNFa, and GM-CSF. These data were indicative of the effective in vitro activity and function of the C10s-2A-MHly dual TRuC T cells in the presence of the CD70 and MSLN antigens alone and in combination.

[0812] These studies were then extended to test the C10s-2A-MHly dual TRuC in alternative target cell lines (N0M0-1, Hs766t and HeLa) expressing low to moderate levels of both the MSLN and CD70 antigens. Mono TRuCs MHly, CIOs, MHls and non-transfected T cells were included for comparison. In the cytotoxicity assay, cell killing efficiency of the MHls, CIOs, or MHly mono TRuC or C10s-2A-MHly dual TRuC T cells was approximately equivalent against HeLa and Hs766t cell lines. TRuC T cells expressing either the CIOs mono TRuC or the C10s-2A-MHly dual TRuC generated the highest cytotoxicity in the N0M0-1 cells, with the mono TRuC showing slightly greater efficiency in this assay.

[0813] Cytokine release was also assessed in the supernatants of each of the aforementioned cocultures. In HeLa cells, the C10s-2A-MHly dual TRuC T cells produced more cytokines (IFNy, IL-2, TNFa, and GM-CSF) than the mono TRuC MHly, CIOs, or MHls T cells, while in both the Hs766t and N0M0-1 cells, the C10s-2A-MHly dual TRuC and mono TRuC MHly, CIOs, or MHls T cells showed generally comparable cytokine release.

[0814] Taken together, these findings indicated that the C10s-2A-MHly dual TRuC successfully targeted both CD70 and MSLN in these in vitro assays.

Example 3: Selection of TCR subunit

[0815] To test the effect of the TCR subunit on MSLN targeting activity and efficiency, two constructs linking the MH1 MSLN binder to different TCR subunits (gamma or epsilon) were directly compared. Dual TRuCs C10s-2A-MHly (SEQ ID NO: 1003) and C10s-2A-MHls (SEQ ID NO: 1004) were evaluated against MHly, CIOs, or MHls mono TRuC (SEQ ID NO: 1001, SEQ ID NO: 1002, SEQ ID NO: 195) and non-transfected controls by flow cytometry and in T cell expansion, cytotoxicity and cytokine release assays.

[0816] The T cell expansion assay showed substantially similar fold expansion over time across all conditions, including the non-transfected control. These data demonstrated that transduction of T cells with either mono or dual TRuCs did not substantially impact the rate of cell growth in vitro. Analysis by flow cytometry showed successful transduction of CIOs, MHly, and MHls mono TRuCs and C10s-2A-MHly and C10s-2A-MHls dual TRuCs as determined by detection of the CD70 and MSLN antigen binders. Flow cytometry data are shown in Figure 4.

[0817] The in vitro efficacy of C10s-2A-MHly and C10s-2A-MHls dual TRuC T cells was tested against MSTO-MSLN, 786-0, OVCAR8 and U20S cell lines and compared to the cytotoxicity of mono TRuC (MHly, CIOs, or MHle) expressing T cells. Data are shown in Figure 5. TRuC T cells expressing either of the C108-2A-MHly and C I Os-2A-MH I s dual TRuCs showed generally similar patterns of cytotoxicity across the 4 cell lines, though C I Os-2A-MH Is dual TRuC T cells generated slightly less against the MSTO-MSLN cell line. Dual TRuC expressing T cells performed much like mono TRuCs having the same target (e.g.„ like the MSLN targeting mono TRuC T cells against the MSTO-MSLN cell line and like the CD70 targeting mono TRuC T cells against the 786-0 cell line). [0818] Assessment of cytokine release in the supernatants of each of the co-cultures outlined above indicated that C10s-2A-MHls dual TRuC T cells generally induced substantially less cytokine release than C108-2A-MHly dual TRuC T cells across all cell types and as measured by levels of IFNy, IL-2, TNFa, and GM-CSF. These data indicated that the MH1 arm of C10e-2A-MH18 may be slightly weaker than that of C108-2A-MHly, perhaps due to competition of the dual CD3s subunits for surface expression.

[0819] To further compare the two dual TRuCs, the memory phenotype of CD4+ or CD8+ transduced T cells was determined, and the proportion of each phenotype was quantified and plotted as shown in Figure 6 and Figure 7. Four phenotypes were used for characterization and were described as Naive (CCR7+CD45R+), Central Memory (CCR7+CD45RA-), Effector memory (CCR7-CD45RA-) and Effector (CCR7-CD45RA+). The resultant immune phenotyping was substantially similar across CD4+ and CD8+ T cells transduced with either C10s-2A-MH18 or ClOs- 2A-MHly dual TRuCs. Likewise, immune phenotyping of T cells transduced with dual TRuCs was similar to that seen for T cells transduced with mono TRuCs.

Example 4: Efficacy of dual TRuC T cells in vivo

[0820] The antitumoral efficacy of T cells expressing CD70/MSLN dual targeting TRuCs will be tested in a xenograft mouse model and compared to the efficacy of T cells expressing either a CD70 or MSLN targeting mono TRuC or non-transduced control T cells.

[0821] Based on the studies above, it is hypothesized that T cells transduced with CD70/MSLN dual targeting TRuCs will successfully target both CD70 and MSLN antigens in vivo and that their antitumoral efficacy will be roughly equivalent or better than the efficacy of T cells transduced with a mono TRuC directed at the same target.

[0822] The antitumoral efficacy of T cells expressing CD70/MSLN dual targeting TRuCs will also be tested in a dual -tumor xenograft mouse model, in which both CD70+ and MSLN+ tumors are established in each mouse prior to administration of CD70/MSLN dual targeting TRuCs, CD70 mono targeting TRuCs, or MSLN mono targeting TRuCs, or vehicle or non-transduced T cell controls. Based on the studies provided herein, it is hypothesized that T cells transduced with certain CD70/MSLN dual targeting TRuCs will successfully target both CD70 and MSLN expressing tumors concurrently in in vivo. Example 5: Efficacy of dual TRuC T cells in vivo

[0823] The antitumoral efficacy of T cells expressing CD70/MSLN dual targeting TRuCs, as compared to the efficacy of T cells expressing either a CD70 mono targeting TRuC or a MSLN mono targeting TRuC, was tested in a xenograft mouse model. MSLN or CD70 expressing tumors were established in NSG mice by s.c. administration of MSTO-MSLN or 786-0 tumor cells, respectively. On Day 0, mice were administered vehicle control, non-transduced (NT) control T cells, CD70 targeting TRuCs with the CD70 binder on the CD3 epsilon chain (CIOs), MSLN targeting TRuCs with the MSLN binder on the CD3 gamma chain (MHly), MSLN targeting TRuCs with the MSLN binder on the CD3 epsilon chain (TC-210), or C108-T2A-MHly dual targeting TRuCs (which have the CD70 binder on the epsilon chain and the MSLN binder on the gamma chain). The results are provided in Figures 8A and 8B. In vivo efficacy of the dual TRuCs was observed against both tumor types, confirming that T cells transduced with the CD70/MSLN dual targeting TRuCs successfully targeted both CD70 and MSLN antigens in vivo. The dual TRuC was at least as effective as the mono MSLN targeting TRuCs against MSLN expressing tumors (Figure 8A), and was more effective than the CD70 mono targeting TRuC against CD70 expressing tumors (Figure 8B).

[0824] In a separate study, the same control groups and mono TRuCs (CIOs, MHly, and TC-210) were compared to C10a-T2A-MHly as well as the dual TRuC format C 10s-T2A-MH I s. in which the CD70 and MSLN binders are each linked to an epsilon chain. First, an in vitro study confirmed the two dual TRuCs exhibited similar expansion overtime (Figure 9A). Next, the MSTO-MSLN s.c. tumor model was used to assess in vivo efficacy of these two dual TRuCs. The tumor volume over time was measured following administration of the indicated TRuCs to mice bearing MSLN expressing tumors. The results showed that while C10a-T2A-MHly dual TRuCs again showed comparable efficacy against MSLN expressing tumors as MSLN targeting mono TRuCs, the dual TRuC in the C10e-T2A-MH18 format only delayed MSLN expressing tumor growth (Figure 9B).

Example 6. In vitro testing of additional dual TRuCs

[0825] To investigate the additional CD70/MSLN dual TRuC formats described above and shown in Figure 1 and Table 2, T cell expansion, TRuC expression, cytotoxicity, and cytokine release assays were performed. Dual TRuCs C10s-2A-MHly (SEQ ID NO: 1003), C10s-2A-MHly Optil (SEQ ID NO: 1012), C10s-2A-MHly Opti2 (SEQ ID NO: 1013), C10-MH1 tandem (SEQ ID NO: 1014), MH1- C10 tandem (SEQ ID NO: 1015), and ClOy -2A-MH18 (SEQ ID NO: 1016) were generated using cells from at least two different donors and evaluated.

[0826] The T cell expansion assay showed that all of the dual TRuCs generated from each donor expanded (representative donor shown in Figure 10), demonstrating that transduction of T cells with any of these dual TRuCs did not substantially impact the rate of cell growth in vitro. Analysis by flow cytometry for MH1 and CIO expression is provided in Figure 11 and Figure 12. Figure 11 shows that there was successful expression of MH1 and CIO in each of the dual TRuCs, except for C10s-2A- MHly Optil, which did not successfully express either arm of the TRuC. The MFI for the MH1 portion of the TRuC is indicated below each flow plot. In contrast to the Optil version of C 1 Os-2 A - MHly, C10e-2A-MHly Opti2 exhibited improved surface detection of MH1 compared to C10e-2A- MHly without codon optimization. Figure 12 provides the MFI for CIO and shows that there was successful expression of CIO in each group tested (C10s-2A-MHly Optil, which failed expression as shown in Figure 11, was not included in this experiment). CIO MFI was highest in the C10e-2A- MHly Opti2 and C10-MH1 tandem groups.

[0827] Next, the in vitro efficacy of CD70/MSLN dual TRuCs C10a-T2A-MHly, C10a-2TA-MHly Opti2, C10-MH1 tandem, MH1-C10 tandem, and C10y-T2A-MHle was tested against 786-0 (high CD70 expression, no MSLN expression), MSTO-MSLN (high MSLN expression, no CD70 expression), OVCAR8 (high MSLN expression and moderate expression of CD70), and U2OS (moderate expression of both CD70 and MSLN) cell lines. Results for cells from a representative donor are provided in Figure 13 and show that each of the tested dual TRuCs exhibited comparable cytotoxicity.

[0828] Cytokine release (IFNy, IL-2, GM-CSF, and TNFa) was measured in the supernatants of each of the co-cultures outlined above for Figure 13, and results from two representative donors are provided in Figures 14-17. Figure 14A and Figure 14B provide the cytokine release results in response to MSTO-MSLN cells for Donor A and Donor B TRuCs, respectively. Figure 15A and Figure 15B provide the cytokine release results in response to 786-0 cells for Donor A and Donor B TRuCs, respectively Figure 16A and Figure 16B provide the cytokine release results in response to 0VCAR8 cells for Donor A and Donor B TRuCs, respectively. Figure 17A and Figure 17B provide the cytokine release results in response to U2OS cells for Donor A and Donor B TRuCs, respectively. Each dual TRuC produced a cytokine response against tumors expressing MSLN and not CD70, tumors expressing CD70 and not MSLN, and tumors expressing both antigens, with the exception of the C10-MH1 tandem dual TRuC, which exhibited little or no cytokine production in response to cells with moderate MSLN expression.

[0829] To further assess the effectiveness of the MH1 arm of the tandem dual TRuCs, cells were generated from an additional donor and tested for cytokine responses against MSTO-MSLN cells (high MSLN expression, no CD70 expression). In this set of experiments, MSTO-MSLN cells were co-cultured with dual TRuCs C10e-2A-MHly, C10e-2A-MHly Opti2, C10-MH1 tandem, and MH1- C10 tandem; and with mono TRuCs CIOs and MHly. The results are provided in Figure 18. The C10-MH1 tandem dual TRuC did not produce cytokine in response to MSTO-MSLN cells (levels similar to the CIOs mono TRuC which does not include an anti-MSLN binder). The data suggested that the MH1 (MSLN binding) arm of the C10-MH1 tandem dual TRuC was less effective against MSLN expressing cells, but confirmed that the remaining dual TRuCs tested exhibited production of IFNy, IL-2, GM-CSF, and TNFa in response to high MSLN expressing cells.

[0830] An additional set of experiments was conducted to assess the effect of using a P2A linker sequence or P2A with a furin cleavage site (fP2A), rather than a T2A linker sequence as in the dual constructs tested in the preceding experiments; and to compare C10s-2A-MHly dual TRuCs to dual TRuCs expressing CIO on the epsilon chains and two MH1 binding domains on the gamma chain (C108-2A-(MHl_MHl)y, i.e., the fourth construct from the left in Figure 1). As a control, a mono TRuC design with two MH1 binding domains on the gamma chain and no CIO arm was also included. Binder expression for each of the constructs is shown in Figure 19. C108-T2A-MH1Y and ClOs- P2A-MHly exhibited high expression of both the CIO and MH1 arms; in contrast, C108-fP2A-MHly exhibited much lower expression of both arms and C10S-T2A-(MH1_MH1)Y exhibited expression of the CIO arm only. C10S-T2A-MH1Y, C10S-P2A-MH1Y, C10S-T2A-(MH1_MH1)Y dual TRuCs and mono TRuCs MHl-MHly, CIOs, and MHly were incubated with 786-0 (high CD70 expression, no MSLN expression), MSTO-MSLN (high MSLN expression, no CD70 expression), OVCAR8 (high MSLN expression and moderate expression of CD70), or U2OS (moderate expression of both CD70 and MSLN) cell lines to determine the cytotoxicity of each construct against target cells. The lysis assays confirmed that the C10S-T2A-(MH1_MH1)Y expressing cells did not significantly lyse cells expressing high levels of MSLN with no CD70 expression (MSTO-MSLN), and exhibited lower lysis of cells with high levels of MSLN and moderate levels of CD70 expression (OVCAR8) (Figure 20). A similar trend was observed with cytokine production from each of the cell lines (786-0, MSTO- MSLN, 0VCAR8, and U2OS, Figures 21A, 21B, 21C, and 21D, respectively). Notably, C10s-T2A- (MH1_MH1)Y exhibited little or no cytokine production in response to MSTO-MSLN and 0VCAR8 cell lines, whereas C108-T2A-MH1Y and C108-P2A-MH1Y produced IFNy, IL-2, GM-CSF, and TNFa in response to each cell line.

[0831] Together, the data showed that only certain dualCD70/MSLN dual TRuC formats were fully functional in vitro. Accordingly, further studies were conducted to further assess the dual TRuCs that were highly functional in vitro, i.e., C10S-2A-MH1Y, C108-2A-MH1Y Opti2, MH1-C10 tandem, and ClOy -2A-MH18 (each schematically depicted in Figure 22; sequences are provided in Table 2). The cytokine response to MSTO-MSLN and 786-0 cells of these four dual TRuCs generated from a third representative donor is shown in Figure 23A (MSTO-MSLN cells) and Figure 23B (786-0 cells) and confirmed that each dual TRuC exhibited a cytokine response to both cell lines.

[0832] The memory phenotype of CD4+ (Figure 24) and CD8+ (Figure 25) transduced T cells showing the most promise based on the studies above (C108-2A-MH1Y, C108-2A-MH1Y Opti2, MH 1 -CIO tandem, and ClOy -2A-MHle) was determined, and the proportion of each phenotype was quantified and plotted. Transduced T cells were stained for CD45RA and CCR7 to determine the proportion of Naive (CCR7+CD45R+), Central Memory (CCR7+CD45RA-), Effector memory (CCR7-CD45RA-) and Effector (CCR7-CD45RA+) in each group. No major difference in T cell differentiation phenotype was identified among the dual TRuCs.

Example 7. Efficacy of additional dual TRuC T cells in vivo

[0833] Based on the studies above, the four constructs C10e-T2A-MHlg; C10e-T2A-MHlg (codon optimization 2; “Opti2”); C10g-T2A-MHle; and (MHl-ClO)e (“MH1 and CIO tandem”) were selected for additional in vivo experiments. [0834] The antitumoral efficacy of the four CD70/MSLN dual targeting TRuCs listed above in comparison to CD70 mono targeting or MSLN mono targeting TRuCs were tested in the xenograft mouse model described above in Example 5. For mono targeting TRuCs in this experiment, CIO was administered only to mice bearing CD70 expressing tumors and TC-210 was administered only to mice bearing MSTO-MSLN expressing tumors; vehicle, NT, and all dual TRuC groups were tested in both sets of mice. The results of the study are provided in Figure 26. Each of the dual TRuCs was more effective against 786-0 (CD70 expressing) tumors as compared to the CD70 mono targeting TRuC (CIO) (left panel). In mice harboring MSLN expressing tumors (MSTO-MSLN), each of the dual TRuCs was effective; however, improved in vivo efficacy was observed for the C10e-T2A- MHlg-Opti2, C10g-T2A-MHle, and (MHl-ClO)e tandem dual TRuCs as compared to C10e-T2A- MHlg dual TRuCs (right panel).

Table 1. Exemplary Antigen binding domain sequences

Table 2. Exemplary construct sequences

Table 3. Exemplary sequences Table 4: Exemplary PD-1 Switch Molecule Sequences

Table 5: Examples of Anti-PD-1 Antibody Sequences and Fusion Protein Sequences

Table 6: Examples of IL-15 and IL-15R Sequences

OTHER EMBODIMENTS

[0835] The disclosure set forth above may encompass multiple distinct inventions with independent utility. Although each of these inventions has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the inventions includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and/or properties as described herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed in this application, in applications claiming priority from this application, or in related applications. Such claims, whether directed to a different invention or to the same invention, and whether broader, narrower, equal, or different in scope in comparison to the original claims, also are regarded as included within the subject matter of the inventions of the present disclosure.

[0836] While preferred embodiments of the present disclosure 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 present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.