WO2023091420A2 - Compositions and methods for t cell engineering - Google Patents

Abstract

Provided herein are recombinant nucleic acids encoding a T cell receptor (TCR) fusion protein (TFP), modified T cells expressing the encoded molecules, and methods of use thereof for the treatment of diseases, including cancer. The modified T cells can comprise a functional disruption of an endogenous gene encoding a TCR chain and/or encoding a major histocompatibility complexes (MHC) molecule or a subunit thereof.

Description

COMPOSITIONS AND METHODS FOR T CELL ENGINEERING

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/279,922, filed November 16, 2021, U.S. Provisional Application No. 63/328,903, filed April 8, 2022, and U.S. Provisional Application No. 63/351,637, filed June 13, 2022, the entire content of each of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Most patients 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] Human T cell therapies rely on enriched or modified human T cells to target and kill cancer cells in a patient. To increase the ability of T cells to target and kill a particular cancer cell, methods have been developed to engineer T cells to express constructs which direct T cells to a particular target cancer cell. Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs), which comprise binding domains capable of interacting with a particular tumor antigen, allow T cells to target and kill cancer cells that express the particular tumor antigen.

[0004] Besides the ability of genetically modified T cells expressing a CAR or an engineered TCR to recognize and destroy respective target cells in vitro/ex vivo, successful patient therapy with engineered T cells may require the T cells to be capable of strong activation, expansion, persistence over time, effective tumor targeting, and, in case of relapsing disease, enabling a ‘memory’ response.

[0005] Whilst not wishing to be bound by theory, typically T cell therapies may require collection of T cells from a patient, preparation, and subsequent return to the patient’s system. The need exists for “off the shelf’ T cell therapies that can be provided to a patient in need without first harvesting the T cells for modification. In this case, however, there can arise host rejection concerns.

SUMMARY OF THE INVENTION

[0006] A need exists for modified T cells with reduced risk of host response and maintained tumor targeting efficiency. In addition, there is a 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. The genetically engineered (e.g., modified) cells provided herein can comprise a T-cell receptor fusion protein (TFP) or a recombinant nucleic acid sequence encoding the TFP, and a functional disruption of one or more endogenous genes encoding TCR subunits such as TCR alpha chain and TCR beta chain. The genetically engineered cells provided herein can comprise a functional disruption of an endogenous gene encoding a major histocompatibility complexes (MHC) molecule or a subunit thereof. For example, the genetically engineered cells provided herein can comprise a functional disruption of an endogenous gene encoding a beta-2 -microglobulin (B2M) molecule. The genetically engineered cells provided herein can further comprise an enhancing agent that enhances persistence of the cells, and/or can further comprise an agent that protects the cells from NK cell-mediated lysis.

[0007] In an aspect, the present disclosure provides a modified cell comprising a recombinant nucleic acid comprising (I) a first sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (II) an antibody domain comprising an antigen binding domain; and wherein the TCR subunit and the antibody domain are operatively linked, wherein the TFP functionally incorporates into an endogenous TCR complex when expressed in the modified cell, wherein the modified cell comprises a functional disruption of an endogenous major histocompatibility complex (MHC) molecule, wherein the modified cell comprises an enhancing agent or a sequence encoding the enhancing agent that enhances persistence of the modified cell, and wherein the enhancing agent comprises an interleukin- 15 (IL- 15) polypeptide or a fragment thereof. In some embodiments, the endogenous MHC molecule comprises all endogenous MHC molecules within the modified cell.

[0008] In an aspect, the present disclosure provides a modified cell comprising a recombinant nucleic acid comprising (I) a first sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (II) an antibody domain comprising an antigen binding domain; and wherein the TCR subunit and the antibody domain are operatively linked, wherein the TFP functionally incorporates into an endogenous TCR complex when expressed in the modified cell; wherein the modified cell comprises a functional disruption of an endogenous major histocompatibility complex (MHC) molecule; and wherein the modified cell comprises an agent, or a sequence encoding the agent, that inhibits NK cell activity against the modified cell. In some embodiments, the NK cell activity is NK-cell mediated lysis.

[0009] In some embodiments, the agent comprises an HLA-E and/or HLA-G polypeptide. In some embodiments, the agent is a B2M-HLA-E or B2M-HLA-G fusion protein. In some embodiments, the agent is a heterodimer comprising a B2M fused to HLA-E or HLA-G. In some embodiments, the HLA-E is HLA- E*01:03. In other embodiments, the HLA-E is HLA-E*01:01. In some embodiments, the B2M is fused to the HLA-E or HLA-G via a Gly-Ser linker. For example, in some embodiments, the linker linking the B2M to the HLA-E or HLA-G comprises a G4S sequence (e.g., (G4S)n, wherein n is an integer from 1-10). In some embodiments, the linker linking the B2M to the HLA-E or HLA-G comprises (G4S)3 or (G4S)4. In some embodiments, the B2M is a mutated B2M. In some embodiments, the B2M comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 420. In some embodiments, the B2M comprises an amino acid sequence according to SEQ ID NO: 420. In some embodiments, the agent further comprises a HLA-G leader peptide sequence. Thus, in some embodiments, the agent is a heterotrimer comprising a B2M fused to the HLA-E (e.g., HLA-E*01:03) and the HLA-G leader peptide. In some embodiments, the HLA-G leader peptide comprises a sequence according to SEQ ID NO: 418. In some embodiments, the HLA-G leader peptide is fused to the B2M via a Gly-Ser linker. For example, in some embodiments, the linker linking the HLA-G leader peptide to the B2M comprises a G4S sequence (e.g., (G4S)n, wherein n is an integer from 1-10). In some embodiments, the linker linking the HLA-G leader peptide to the B2M comprises (G4S)3 or (G4S)4. In some embodiments, the agent comprises an HLA-G binding protein, a linker, a mutated B2M, a second linker, and an HLA-E*01:03.

[0010] In some embodiments, sequence encoding the agent comprises a B2M signal sequence. In some embodiments, the agent comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 423. In some embodiments, the agent comprises a sequence according to SEQ ID NO: 423.

[0011] In some embodiments, the recombinant nucleic acid is linked to the sequence encoding the agent that inhibits NK cell activity by a cleavable linker. In some embodiments, the cleavable linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A cleavage site.

[0012] In some embodiments, wherein the recombinant nucleic acid further comprises a sequence encoding a signal peptide. In some embodiments, the signal peptide is a GM-CSF signal peptide. In some embodiments, the recombinant nucleic acid molecule further comprises a sequence encoding a protease. In some embodiments, the protease is a furin.

[0013] In some embodiments, the sequence encoding the agent is contained within a different recombinant nucleic acid molecule than the recombinant nucleic acid molecule containing the first and second sequences. In some embodiments, the first sequence, the second sequence, and the sequence encoding the agent are contained within the same recombinant nucleic acid molecule. In some embodiments, the recombinant nucleic acid molecule encodes, from N-terminus to C-terminus, a GM-CSF signal peptide operatively linked to an antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to a furin, operatively linked to a linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional antigen binding domain, operatively linked to a TCR gamma constant domain, operatively linked to a linker, operatively linked to a T2A sequence, operatively linked to a B2M leader sequence, operatively linked to an HLA-G binding peptide, operatively linked to a linker, operatively linked to a mutB2M, operatively linked to a linker, operatively linked to HLA-E*01:03.

[0014] In some embodiments, the recombinant nucleic acid molecule encodes, from N-terminus to C- terminus, a GM-CSF signal peptide operatively linked to an antigen binding domain, operatively linked to a TCR gamma constant domain, operatively linked to a furin, operatively linked to a linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to a linker, operatively linked to a T2A sequence, operatively linked to a B2M leader sequence, operatively linked to an HLA-G binding peptide, operatively linked to a linker, operatively linked to a mutB2M, operatively linked to a linker, operatively linked to HLA-E*01:03.

[0015] In some embodiments, the recombinant nucleic acid molecule encodes, from N-terminus to C- terminus, a B2M signal peptide operatively linked to an HLA-G binding peptide, operatively linked to a linker, operatively linked to a mutB2M, operatively linked to a linker, operatively linked to HLA-E*01:03, operatively linked to a T2A sequence, operatively linked to a GM-CSF signal peptide, operatively linked to an antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to a furin, operatively linked to a linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional antigen binding domain, operatively linked to a TCR gamma constant domain.

[0016] In some embodiments, the recombinant nucleic acid molecule encodes, from N-terminus to C- terminus, a B2M signal peptide operatively linked to an HLA-G binding peptide, operatively linked to a linker, operatively linked to a mutB2M, operatively linked to a linker, operatively linked to HLA-E*01:03, operatively linked to a T2A sequence, operatively linked to a GM-CSF signal peptide, operatively linked to an antigen binding domain, operatively linked to a TCR gamma constant domain, operatively linked to a furin, operatively linked to a linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional antigen binding domain, operatively linked to a TCR delta constant domain.

[0017] In some embodiments, the modified cell comprises a functional disruption of an endogenous MHC molecule, wherein the endogenous MHC molecule comprises an MHC class I molecule, a MHC class II molecule, or a combination thereof. In some embodiments, the endogenous MHC molecule comprises all endogenous MHC molecules within the modified cell. In some embodiments, the functional disruption of the MHC molecule comprises inactivating a gene encoding the MHC molecule or subunit thereof. In some embodiment, inactivating the gene encoding the MHC molecule or subunit thereof comprises knocking out or knocking down the gene. In some embodiment, the gene encoding the MHC molecule or subunit thereof comprises a gene encoding a B2M molecule. In some embodiments, the modified cell does not express any endogenous MHC molecules on a surface of the modified cell.

[0018] In some embodiments, the modified cell comprises a functional disruption of an endogenous TCR chain selected from TCR alpha and TCR beta. In some embodiments, the modified cell comprises a functional disruption of the TCR alpha and the TCR beta chains. In some embodiments, the functional disruption is a disruption of a gene encoding the endogenous TCR chain. In some embodiments, the disruption of a gene encoding the endogenous TCR chain is a removal of a sequence of the gene encoding the endogenous TCR chain from the genome of the modified cell.

[0019] In some embodiments, the TFP further comprises a TCR intracellular domain. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta. In some embodiments, all three of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta. In some embodiments, the TCR transmembrane domain, and the TCR intracellular domain do not comprise a variable domain of TCR delta. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain do not comprise a variable domain of TCR gamma.

[0020] In some embodiments, the endogenous MHC molecule comprises an MHC class I molecule, a MHC class II molecule, or a combination thereof. In some embodiments, the functional disruption of the MHC molecule comprises inactivating a gene encoding the MHC molecule or subunit thereof. In some embodiments, inactivating the gene encoding the MHC molecule or subunit thereof comprises knocking out or knocking down the gene. In some embodiments, the gene encoding the MHC molecule or subunit thereof comprises a gene encoding a beta-2 -microglobulin (B2M) molecule. In some embodiments, the modified cell does not express any MHC molecules on a surface of the modified cell. In some embodiments, the TFP further comprises a TCR intracellular domain.

[0021] In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR alpha. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR beta. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma. In some embodiments, all three of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma.

[0022] In some embodiments, the recombinant nucleic acid comprising a second sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain or a TCR delta constant domain, or a second sequence encoding a TCR gamma constant domain and a TCR delta constant domain.

[0023] In some embodiments, the second sequence further encodes a TCR transmembrane domain, wherein the TCR transmembrane domain is a TCR gamma transmembrane domain or a TCR delta transmembrane domain. In some embodiments, the first sequence and the second sequence are contained in a same recombinant nucleic acid molecule. In some embodiments, the recombinant nucleic acid molecule further comprises a sequence encoding a protease cleavage site. In some embodiments, the first sequence and the second sequence are contained in two separate recombinant nucleic acid molecules. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR alpha. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR alpha. In some embodiments, the constant domain of TCR alpha is murine. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain further comprise a TCR alpha transmembrane domain and a TCR alpha intracellular domain. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain do not comprise a variable domain of TCR alpha. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR beta. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR beta. In some embodiments, the constant domain of TCR beta is murine. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain further comprise a TCR beta transmembrane domain and a TCR beta intracellular domain. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain do not comprise a variable domain of TCR beta. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR gamma. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain further comprise a TCR gamma transmembrane domain and a TCR gamma intracellular domain. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain do not comprise a variable domain of TCR gamma. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR delta. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain further comprise a TCR delta transmembrane domain and a TCR delta intracellular domain. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain do not comprise a variable domain of TCR delta. In some embodiments, the TCR delta or the TCR delta constant domain comprises a sequence of SEQ ID NO: 243. In some embodiments, the TCR gamma or the TCR gamma constant domain comprises a sequence of SEQ ID NO: 21.

[0024] In some embodiments, the modified cell comprises the enhancing agent. In some embodiments, the modified cell comprises the sequence encoding the enhancing agent. In some embodiments, the recombinant nucleic acid molecule comprises a third sequence that is the sequence encoding the enhancing agent. In some embodiments, the first sequence and the third sequence are operatively linked by a first linker. In some embodiments, the first linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A cleavage site. In some embodiments, the IL-15 polypeptide is secreted. In some embodiments, the IL-15 polypeptide comprises a sequence of SEQ ID NO: 385. In some embodiments, the third sequence further encodes an IL-15 receptor (IL-15R) subunit or a fragment thereof. In some embodiments, the IL-15R subunit is IL-15R alpha (IL-15Ra). In some embodiments, IL-15 and IL-15Ra are operatively linked by a second linker. In some embodiments, the second linker is not a cleavable linker. In some embodiments, the second linker comprises 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 an integer from 1 to 4. In some embodiments, n is 3. In some embodiments, the second linker comprises a sequence of SEQ ID NO: 378 or 405. In some embodiments, the third sequence encodes a fusion protein comprising the IL- 15 polypeptide linked to the IL-15Ra subunit. In some embodiments, the IL-15 polypeptide is linked to N-terminus of the IL-15Ra subunit. In some embodiments, the fusion protein comprises amino acids 30 - 162 of IL-15. In some embodiments, the fusion protein comprises amino acids 31 - 267 of IL-15Ra. In some embodiments, the fusion protein further comprises a sushi domain. In some embodiments, the fusion protein comprises a sequence of SEQ ID NO: 389. In some embodiments, the fusion protein comprises a sequence of SEQ ID NO: 371. In some embodiments, the fusion protein is expressed on cell surface of the modified cell. In some embodiments, the fusion protein is secreted.

[0025] In another aspect, the present disclosure provides a modified cell comprising a recombinant nucleic acid comprising (I) a first sequence encoding a T cell receptor (TCR) fusion protein (TEP) comprising a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, and (2) a TCR transmembrane domain, and (II) an antibody domain comprising an antigen binding domain; and a second sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain or a TCR delta constant domain, or a second sequence encoding a TCR gamma constant domain and a TCR delta constant domain; wherein the TCR subunit and the antibody domain are operatively linked, wherein the TFP functionally incorporates into an endogenous TCR complex when expressed in the modified cell, and wherein the modified cell comprises a functional disruption of an endogenous major histocompatibility complex (MHC) molecule.

[0026] In some embodiments, the modified cell comprises an enhancing agent or a sequence encoding the enhancing agent that enhances persistence of the modified cell. In some embodiments, the modified cell comprises the enhancing agent. In some embodiments, the modified cell comprises the sequence encoding the enhancing agent. In some embodiments, the recombinant nucleic acid molecule comprises a third sequence that is the sequence encoding the enhancing agent, and wherein the enhancing agent comprises an interleukin- 15 (IL- 15) polypeptide or a fragment thereof. In some embodiments, the first sequence and the third sequence are operatively linked by a first linker. In some embodiments, the first linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A cleavage site. In some embodiments, the IL- 15 polypeptide is secreted. In some embodiments, the third sequence further encodes an IL- 15 receptor (IL-15R) subunit or a fragment thereof. In some embodiments, the IL-15R subunit is IL-15R alpha (IL-15Ra). In some embodiments, IL-15 and IL-15Ra are operatively linked by a second linker. In some embodiments, the second linker is not a cleavable linker. In some embodiments, the second linker comprises 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 an integer from 1 to 4. In some embodiments, n is 3. In some embodiments, the third sequence encodes a fusion protein comprising the IL- 15 polypeptide linked to the IL-15Ra subunit. In some embodiments, the IL- 15 polypeptide is linked to N-terminus of the IL-15Ra subunit.

[0027] In some embodiments, the endogenous MHC molecule comprises all endogenous MHC molecules within the modified cell.

[0028] In some embodiments, the endogenous MHC molecule comprises an MHC class I molecule, a MHC class II molecule, or a combination thereof.

[0029] In some embodiments, the functional disruption of the MHC molecule comprises inactivating a gene encoding the MHC molecule or subunit thereof.

[0030] In some embodiments, inactivating the gene encoding the MHC molecule or subunit thereof comprises knocking out or knocking down the gene.

[0031] In some embodiments, the gene encoding the MHC molecule or subunit thereof comprises a gene encoding a beta-2 -microglobulin (B2M) molecule. In some embodiments, the modified cell does not express any MHC molecules on a surface of the modified cell.

[0032] In some embodiments, the TCR extracellular domain and the TCR transmembrane domain are from a same subunit. In some embodiments, the same subunit is TCR gamma or TCR delta. In some embodiments, the TCR subunit further comprises a TCR intracellular domain. In some embodiments, the TCR intracellular domain is from TCR gamma or TCR beta. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain and the TCR intracellular domain are from a same subunit. In some embodiments, the second sequence further encodes a second antibody domain comprising a second antigen binding domain. In some embodiments, the second antigen binding domain and the antigen binding domain are the same. In some embodiments, the first sequence and the second sequence are contained within the same recombinant nucleic acid molecule. In some embodiments, the first sequence and the second sequence are contained within two different recombinant nucleic acid molecules.

[0033] In some embodiments, the antibody domain is an antibody fragment. In some embodiments, the antibody fragment is a scFv, a single domain antibody domain, a VH domain or a VL domain. [0034] In some embodiments, an antigen binding domain is selected from a group consisting of an anti- mesothelin (MSLN) binding domain, an anti-CD70 binding domain, an anti-Nectin-4 binding domain, and an anti-GPC3 binding domain.

[0035] In some embodiments, the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO:60, a CDR2 of SEQ ID NO: 61, and a CDR3 of SEQ ID NO: 62.

[0036] In some embodiments, the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO:63, a CDR2 of SEQ ID NO : 64, and a CDR3 of SEQ ID NO : 65.

[0037] In some embodiments, the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO:66, a CDR2 of SEQ ID NO:67, and a CDR3 of SEQ ID NO:68.

[0038] In some embodiments, the anti-MSLN binding domain comprises a sequence with at least about 80% sequence identity to a sequence of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71.

[0039] In some embodiments, the TCR subunit and the antibody domain are operatively linked by a linker. [0040] In some embodiments, the linker comprises a sequence of SEQ ID NO: 387.

[0041] In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a signal peptide. In some embodiments, the signal peptide is a GM-CSF signal peptide. In some embodiments, the recombinant nucleic acid molecule further comprises a sequence encoding a protease. In some embodiments, the protease is a furin. In some embodiments, the recombinant nucleic acid comprises a sequence of SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 400, SEQ ID NO: 402, and/or SEQ ID NO: 404. In some embodiments, the recombinant nucleic acid molecule comprises a sequence encoding SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 69, SEQ ID NO: 243, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, and/or SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid molecule encodes, from N-terminus to C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional anti- MSLN antigen binding domain, operatively linked to a TCR gamma constant domain. In some embodiments, the recombinant nucleic acid comprises a sequence of SEQ ID NO: 407, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 404, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, and/or SEQ ID NO: 411. In some embodiments, the recombinant nucleic acid molecule comprises a sequence encoding SEQ ID NO: 366, SEQ ID NO: 362, SEQ ID NO: 69, SEQ ID NO: 243, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 385, SEQ ID NO: 405, and/or SEQ ID NO: 403. In some embodiments, the recombinant nucleic acid molecule encodes, from N-terminus to C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional anti- MSLN antigen binding domain, operatively linked to a TCR gamma constant domain, operatively linked to a linker, operatively linked to a T2A sequence, operatively linked to a IL- 15 polypeptide, operatively linker to a linker, operatively linked to a hIL-15Ra polypeptide. In some embodiments, the recombinant nucleic acid comprises a sequence of SEQ ID NO: 412, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 413, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 414, and/or SEQ ID NO: 404. In some embodiments, the recombinant nucleic acid molecule encodes a sequence of SEQ ID NO: 367, SEQ ID NO: 362, SEQ ID NO: 69, SEQ ID NO: 387, SEQ ID NO: 243, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, and/or SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid molecule encodes, from N-terminus to C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN antigen binding domain, operatively linked to a first linker, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a second linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional anti-MSLN antigen binding domain, operatively linked to a third linker, operatively linked to a TCR gamma constant domain. In some embodiments, the recombinant nucleic acid comprises a sequence of SEQ ID NO: 415, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 413, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 414, SEQ ID NO: 404, SEQ ID NO: 390, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, and/or SEQ ID NO: 411. In some embodiments, the recombinant nucleic acid molecule encodes a sequence of SEQ ID NO: 368, SEQ ID NO: 362, SEQ ID NO: 69, SEQ ID NO: 387, SEQ ID NO: 243, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 385, SEQ ID NO: 405, and/or SEQ ID NO: 403. In some embodiments, the recombinant nucleic acid molecule encodes, from N-terminus to C- terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN antigen binding domain, operatively linked to a first linker, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a second linker, operatively linked to a P2A sequence, operatively linked to another GM- CSF signal peptide, operatively linked to an additional anti-MSLN antigen binding domain, operatively linked to a third linker, operatively linked to a TCR gamma constant domain, operatively linked to a fourth linker, operatively linked to a T2A sequence, operatively linked to a IL- 15 polypeptide, operatively linker to a linker, operatively linked to a hIL-15Ra polypeptide.

[0042] In some embodiments, the modified cell comprises a functional disruption of an endogenous TCR chain. In some embodiments, the endogenous TCR chain 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 embodiments, the endogenous TCR chain that is functionally disrupted has reduced binding to MHC-peptide complex compared to that of an unmodified control cell. In some embodiments, the functional disruption is a disruption of a gene encoding the endogenous TCR chain. In some embodiments, the disruption of a gene encoding the endogenous TCR chain is a removal of a sequence of the gene encoding the endogenous TCR chain from the genome of the modified cell.

[0043] In some embodiments, the modified cell is a T cell. [0044] In some embodiments, the T cell is a human T cell selected from CD4 cells, CD8 cells, naive T-cells, memory stem T-cells, central memory T- cells, double negative T-cells, effector memory T-cells, effector T- cells, ThO cells, TcO cells, Thl cells, Tel cells, Th2 cells, Tc2 cells, Th 17 cells, Th22 cells, alpha/beta T cells, gamma/delta T cells, natural killer (NK) cells, natural killer T (NKT) cells, hematopoietic stem cells and pluripotent stem cells.

[0045] In some embodiments, the T cell is a CD8+ or CD4+ T cell. In some embodiments, the T cell is an allogenic T cell. In some embodiments, the modified cell further comprises 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 embodiments, the inhibitory molecule comprises the first polypeptide comprising at least a portion of PD1 and the second polypeptide comprising a costimulatory domain and primary signaling domain.

[0046] In another aspect, the present disclosure provides a pharmaceutical composition comprising: the modified cell described herein; and a pharmaceutically acceptable carrier.

[0047] In another aspect, the present disclosure provides a method of producing the modified cell described herein, the method comprising functionally disrupting an endogenous MHC molecule of a cell; and transducing the cell containing a functional disruption of the endogenous MHC gene with the recombinant nucleic acid described herein.

[0048] In some embodiments, the method further comprises functionally disrupting an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain; thereby producing a cell containing a functional disruption of an endogenous TCR gene.

[0049] In some embodiments, disrupting the endogenous TCR gene comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets the endogenous gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.

[0050] In some embodiments, disrupting the endogenous MHC molecule comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets an endogenous gene encoding the endogenous MHC molecule.

[0051] In another aspect, the present disclosure provides a method of producing the modified cell described herein, the method comprising transducing a cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid described herein.

[0052] In some embodiments, the cell containing a functional disruption of an endogenous TCR gene is a cell containing a functional disruption of an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.

[0053] In some embodiments, the cell further comprises a functional disruption of an endogenous MHC molecule.

[0054] In some embodiments, the cell comprises a functional disruption of a gene encoding a B2M molecule. [0055] In some embodiments, the cell is a T cell.

[0056] In some embodiments, the T cell is a human T cell. [0057] In some embodiments, the cell containing a functional disruption of an endogenous TCR gene has reduced binding to MHC -peptide complex compared to that of an unmodified control cell.

[0058] In some embodiments, the nuclease protein is a meganuclease, a zinc -finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, or a megaTAL nuclease. [0059] In some embodiments, the sequence comprised by the recombinant nucleic acid is inserted into the endogenous TCR subunit gene at the cleavage site, and wherein the insertion of the sequence into the endogenous TCR subunit gene functionally disrupts the endogenous TCR subunit.

[0060] In some embodiments, the nuclease protein is a meganuclease.

[0061] In some embodiments, the meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence, and wherein the second subunit binds to a second recognition half-site of the recognition sequence.

[0062] In some embodiments, the meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.

[0063] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein.

[0064] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a modified cell produced according to the method described herein; and (b) a pharmaceutically acceptable carrier.

[0065] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a modified cell produced according to the method described herein; and (b) a pharmaceutically acceptable carrier.

[0066] In some embodiments, the modified cell is an allogeneic T cell.

[0067] In some embodiments, less cytokines are released in the subject compared a subject administered an effective amount of an unmodified control cell.

[0068] In some embodiments, less cytokines are released in the subject compared a subject administered an effective amount of a modified cell comprising the recombinant nucleic acid described herein.

[0069] In some embodiments, the method comprises administering the pharmaceutical composition in combination with an agent that increases the efficacy of the pharmaceutical composition.

[0070] In some embodiments, the method comprises administering the pharmaceutical composition in combination with an agent that ameliorates one or more side effects associated with the pharmaceutical composition.

[0071] In some embodiments, the cancer is a solid cancer, a lymphoma or a leukemia. [0072] In some embodiments, the cancer is selected from the group consisting of renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney and stomach cancer.

[0073] In some embodiments, the cancer is associated with low tumor antigen density.

[0074] In some embodiments, less cytokines are released in the subject compared to a subject administered an effective amount of an autologous T cell expressing the TFP described herein.

[0075] In some embodiments, the method does not induce graft versus host disease.

[0076] In some embodiments, the subject has a reduced risk of developing graft versus host disease compared to a subject administered an effective amount of an autologous T cell expressing the TFP described herein.

[0077] In another aspect, the present disclosure provides the modified cell described herein, or the pharmaceutical composition described herein, for use as a medicament or in the preparation of a medicament. [0078] In an aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition provided herein, e.g., a pharmaceutical composition comprising a modified cell provided herein. In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a modified cell produced according to the methods provided herein, and a pharmaceutically acceptable carrier. In some embodiments, the modified cell is an allogeneic T cell. In some embodiments, the modified cell is not derived from the subject. In some embodiments, the cancer is a solid cancer, a lymphoma or a leukemia. In some embodiments, the cancer is selected from the group consisting of mesothelioma, renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, cholangiocarcinoma, pancreatic cancer, kidney and stomach cancer. In some embodiments, the cancer is a cancer associated with mesothelin expression. In some embodiments, the cancer is associated with low tumor antigen density.

[0079] In some embodiments, the method of treatment provided herein does not induce graft versus host disease. In some embodiments, the method of treatment provided herein does not elicit an immune response in the subject against the modified cell. For example, in some embodiments, the method of treatment provided herein does not elicit NK cell lysis activity against the modified cell. In some embodiments, the method provided herein results in reduced NK cell lysis activity against the modified cell, compared to NK cell lysis activity against a modified cell that does not comprise the agent that inhibits NK cell activity. In some embodiments, the modified cells provided herein persist in a subject for a longer period of time compared to modified cells that do not comprise the agent that inhibits NK cell activity. In some embodiments, by employing the methods provided herein, the subject has a reduced risk of rejection of the modified cell compared to a subject administered a modified cell that comprises the TFP and that does not comprise the agent that inhibits NK cell activity. In some embodiments, by employing the methods provided herein, the subject has a reduced risk of NK cell activity against the modified cell compared to a subject administered a modified cell that comprises the TFP and that does not comprise the agent. In an aspect, the present disclosure provides modified cells as described herein, or pharmaceutical compositions comprising the modified cells, for use as a medicament or in the preparation of a medicament.

INCORPORATION BY REFERENCE

[0080] 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

[0081] 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.”, “FIG.”, and “FIGURE” herein) of which: [0082] FIG. 1 shows a schematic of an exemplary double knockout allogeneic T cell.

[0083] F IG. 2 shows flow cytometry data for surface expression of HLA Class I, B2M and/or hTCRa[3 in modified T cells.

[0084] FIG. 3 shows flow cytometry data and related quantification of HLA Class I surface expression and percent knockdown efficiency following B2M knockdown.

[0085] FIG. 4 shows flow cytometry data and related quantification of TFP transduction in modified T cells.

[0086] FIG. 5 shows data from modified T cells tested in a cytotoxicity assay against MSLN expressing cells.

[0087] FIG. 6 shows exemplary schematics of the constructs tested in Example 2.

[0088] FIG. 7 shows data from modified T cells tested in a cytotoxicity assay against MSLN expressing cells.

[0089] FIG. 8 shows in vivo efficacy data for modified T cells.

[0090] FIG. 9 shows data indicating enhanced expansion of allogeneic modified T cells when combined with expression of an IL15fus protein.

[0091] FIG. 10 shows a schematic representation for the generation of allogeneic TRuC T cells and a comparison to an autologous TC-210 TCR.

[0092] FIGs. 11A-D show results of in vitro assays characterizing allogeneic TRuC T cells as compared to autologous. FIG. 11A shows exemplary flow cytometry plots assessing transduction efficiency. FIG. 11B shows stacked bar plots of T cell memory phenotype. FIG. 11C shows results of a luciferase-based cytotoxicity assay after 24hr co-culture with MSTO-MSLN cells. FIG. HD shows the results of a cytokine release assay after 24hr co-culture with MSTO-MSLN cells.

[0093] FIG. 12 shows the in vivo efficacy of allogeneic and autologous TRuC T cells tested in a mouse model.

[0094] FIG. 13 shows the improved in vivo persistence of allogeneic TRuC T cells in tissues collected 19 days after TRuC delivery.

[0095] FIGs. 14A-B show improved in vivo sensitivity and efficacy of allogeneic TRuC T cells against a low density antigen expressing tumor cell line.

[0096] FIGs. 15A-15E show the results of in vivo experiments testing a second gene knock-out in allogeneic TRuC T cells. FIG. 15A shows exemplary flow cytometry plots assessing the gene knock-out(s) in the allogeneic TRuC T cells. FIG. 15B shows stacked bar plots of T cell memory phenotype. FIG. 15C shows results of a luciferase-based cytotoxicity assay after 24hr co-culture with MSTO-MSLN cells. FIG. 15D shows activation markers present after 24, 48 or 96 hours of co-culture with MSTO-MSLN cells. FIG. 15E shows in vivo efficacy of allogeneic TRuC T cells in a mouse model.

[0097] FIG. 16 provides schematic views of exemplary B2M-HLA-E fusion proteins. The picture on the top of the figure shows a fusion including an endogenous B2M signal sequence, an HLA-G leader peptide (also referred to herein as an HLA-G binding protein or HLA-G signal peptide, and the like), a mutated B2M (mB2M), and HLA-E*01:03. The picture on the bottom of the figures provides a schematic view of the structure of a B2M-HLA-E fusion protein, with or without the HLA-G leader peptide (Gomalusse et al., Nat Biotech 35(8)765-772 (2017)).

[0098] FIG. 17 provides flow cytometry data showing that B2M knockout primary T cells transduced with the mB2M-HLA-E fusion protein exhibit high expression of HLA-E.

[0099] FIG. 18 shows that expression of the HLA-E fusion protein protects B2M knockout primary T cells from NK cell mediated cytotoxicity.

[0100] FIG. 19 provides flow cytometry data showing that B2M knockout Jurkat cells transduced with mB2M -HLA-E fusion protein have high expression of HLA-E.

[0101] FIG. 20 shows that expression of the HLA-E fusion protein protects B2M knockout Jurkat cells from NK cell mediated cytotoxicity.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0102] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] As used herein, a “therapeutically effective amount” is the amount of a composition or an active component thereof sufficient to provide a beneficial effect or to otherwise reduce a detrimental non-beneficial event to the individual to whom the composition is administered. By “therapeutically effective dose” herein is meant a dose that produces one or more desired or desirable (e.g. , beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0109] 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.

[0110] The term “stimulation” refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like, [oni] The term “stimulatory molecule” or “stimulatory domain” refers to a molecule or portion thereof expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway. In one aspect, the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or “IT AM”. Examples of an ITAM containing primary cytoplasmic signaling sequence that is of particular use in the invention includes, but is not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”) and CD66d.

[0112] The term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC’s) on its surface. T cells may recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0113] “Major histocompatibility complex (MHC) molecules 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.

[0114] The human leukocyte antigen system (HLA) is the name of 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.

[0115] 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 selfantigens. 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. 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. Other HLA- A alleles of interest are HLA-A*0101, HLA-A*2402, and HLA-A*0301. Widely expressed HLA-B alleles of interest are HLA-B*3501, HLA-B*0702 and HLA-B*3502.

[0116] An “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the TFP containing cell, e.g., a modified T-T cell. Examples of immune effector function, e.g., in a modified T-T cell, include cytolytic activity and T helper cell activity, including the secretion of cytokines. In an embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In an embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.

[0117] A primary intracellular signaling domain can comprise an ITAM (“immunoreceptor tyrosine-based activation motif’). Examples of ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d DAP 10 and DAP 12.

[0118] The term “costimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Costimulatory molecules include but are not limited to an MHC class 1 molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CDl la/CD18) and 4-1BB (CD137). A costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like. The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof. The term “4- IBB” 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” is defined as 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.

[0119] 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.

[0120] The terms “antibody fragment” refers 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 such as sdAb (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments.

[0121] 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. [0122] “Heavy chain variable region” or “VH” 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. A camelid “VHH” domain is a heavy chain comprising a single variable antibody domain.

[0123] Unless specified, as used herein a scFv may have the VL and VH variable 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 VH-linker-VL. In some embodiments, the linker may comprise SEQ ID NO: 401.

[0124] The portion of the TFP composition of the 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), a single chain antibody (scFv) derived from a murine, humanized or human antibody (Harlow et al., 1999, In: Using Antibodies: A Eaboratory 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). In one aspect, the antigen binding domain of a TFP composition of the disclosure comprises an antibody fragment. In a further aspect, the TFP comprises an antibody fragment that comprises a scFv or a sdAb.

[0125] 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.

[0126] 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.

[0127] 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. [0128] Mesothelin (MSLN) refers to a tumor differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. Mesothelin is over expressed in several human tumors, including mesothelioma and ovarian and pancreatic adenocarcinoma.

[0129] The term “interleukin 15” or “IL-15” refers to a pleiotropic cytokine that play important roles in maintenance and homeostatic expansion of various immune cells. IL- 15 plays a critical role in the development of the NK lineage, and in survival, expansion, and function of NK cells. Local secretion of pleiotropic cytokines such as IL- 15 in tumor microenvironment (TME) contributes to enhanced anti -tumor immunity. IL- 15 is also involved in lymphocyte homeostasis as lymphocytes depend upon IL- 15 for survival or expansion. IL-15 also plays multiple roles in peripheral innate and adaptive immune cell functions. IL-15 is trans-presented by antigen presenting cells and has a crucial role in the induction of central memory T cell subset and enhanced cytolytic effectors. It aids in T cell survival by reducing activation induced cell death (AICD). Human IL- 15 precursor protein has two known isoforms based on the length of signal peptide. IL- 15 (also referred to as IL-15-S48AA or IL-15LSP for “long signal peptide”) has a 48 amino acid signal peptide and propeptide while IL-15-S21AA or IL-15SSP (for “short signal peptide”), which is expressed from an alternatively spliced mRNA has a 21 amino acid signal peptide and propeptide. IL-15SSP has been shown not to be secreted, but rather stored intracellularly in the cytoplasm.

[0130] The term “interleukin 15 receptor” or “IL-15R” refers to a receptor complex that IL-15 binds to and signals through. IL-15R is composed of three subunits, IL-15 receptor alpha chain (“IL-15Ra” or CD215), IL- 2 receptor beta chain (“IL-2R[3” or CD 122) and IL-2 receptor gamma/the common gamma chain (“IL-2Ry/yc” or CD132). Human IL-15Ra precursor protein has a 30 amino acid signal peptide, a 175 amino acid extracellular domain, a 23 amino acid single membrane -spanning transmembrane stretch, and a 39 amino acid cytoplasmic (or intracellular) domain and contains N- and O-linked glycosylation sites. IL-15Ra contains a Sushi domain (amino acid 31-95) which is essential for IL-15 binding. IL-15Ra also exists as a soluble form (sIL-15Ra). sIL-15Ra is constitutively generated from the transmembrane receptor through a defined proteolytic cleavage, and this process can be enhanced by certain chemical agents, such as PMA. The human sIL-15Ra, about 42 kDa in size, may could prolong the half-life of IL- 15 or potentiate IL- 15 signaling through IL- 15 binding and IL-2R[3/yc heterodimer. Although IL-15R shares subunits with IL-2Rthat contain the cytoplasmic motifs required for signal transduction, IL- 15 signaling has separate biological effects in vivo apart from many biological activities overlapping with IL-2 signaling due to IL-15Ra subunit that is unique to IL-15R, availability and concentration of IL-15, the kinetics and affinity of IL- 15 -IL- 15 Ra binding. IL-15 binds to IL-15Ra specifically with high affinity, which then associates with a complex composed of IL-2RJ3 and IL-2Ry/yc subunits, expressed on the same cell (“cis-presentation”) or on a different cell (“transpresentation”). The interaction between IL-15 and IL-15Ra is independent of the complex composed of IL- 2R[3 and IL-2Ry/yc subunits. IL- 15 binding to the IL-2R[3/yc heterodimeric receptor induces JAK1 activation that phosphorylates STAT3 via the beta chain, and JAK3 activation that phosphorylates STAT5 via the gamma chain. The IL-15/IL-15R interaction modulates not only T-cell development and homeostasis, but also in memory CD8+ T-cell and NK cell development, maintenance, expansion and activities. [0131] 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.

[0132] The term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0133] The term “allogeneic” or, alternatively, “allogenic,” 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.

[0134] The term “xenogeneic” refers to a graft derived from an animal of a different species.

[0135] 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 and the like.

[0136] 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.

[0137] 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. [0138] The term “effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological or therapeutic result. [0139] The term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0140] The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0141] The term “expression” refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

[0142] The term “functional disruption” 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).

[0143] The term “transfer vector” refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “transfer vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0144] The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. [0145] The term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.

[0146] The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453- 1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR™ gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen, and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0147] The term “homologous” or “identity” refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g. , if half (e.g. , five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.

[0148] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. In general, the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0149] “Human” or “fully human” refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.

[0150] The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0151] 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.

[0152] 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.

[0153] The term “operably linked” or “transcriptional control” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.

[0154] 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. [0155] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double -stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et 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)).

[0156] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0157] The term “promoter” refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. [0158] The term “promoter/regulatory sequence” refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0159] The term “constitutive” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0160] The term “inducible” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

[0161] The term “tissue-specific” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0162] 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 (G4S)_n, wherein n=l to 3.

[0163] 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 capsynthesizing 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.

[0164] As used herein, "/A? 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. [0165] As used herein, a “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA. In the preferred embodiment of a construct for transient expression, the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.

[0166] 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 fortranscription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3’ end at the cleavage site. [0167] 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.

[0168] The term “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.

[0169] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human).

[0170] The term, a “substantially purified” cell refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0171] The term “therapeutic” as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.

[0172] 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, pancreatic cancer, and the like.

[0173] The term “transfected” or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0174] 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. Preferably, the recognition sequence for a meganuclease of the present disclosure is 22 base pairs. A meganuclease can be an endonuclease that is derived from I-Crel and can 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). A meganuclease as used herein 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." Meganucleases of the present disclosure are substantially non-toxic when expressed in cells, particularly in human T cells, such that cells can 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.

[0175] 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. The two meganuclease subunits will generally be non-identical in amino acid sequence and will recognize non-identical DNA sequences. Thus, single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences. 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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, sequence-specific homing endonuclease.

[0180] 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.

[0181] Provided herein are compositions of matter and methods of use for the treatment of a disease such as cancer, using modified T cells comprising a T cell receptors (TCR) fusion protein (TFP) in combination with an IL-15 and/or IL-15Ra polypeptide. Advantageously, when these IL-15 and/or IL-15-Ra proteins are expressed in combination with the TCR fusion proteins, they can confer increased persistence, prolonged activity, and increased efficacy on the T cells for treating the malignancies described 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. 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., CD 137), CD27 and/or CD28.

[0182] As used herein, “NK cell lysis inhibitor” and “agent that inhibits NK cell activity” and the like refer to a peptide or protein (including a fusion protein) that serves as a negative signal to NK cells and/or macrophages and prevents or reduces NK cell activity such as NK cell-mediated lysis against a cell that expresses the peptide or protein.

[0183] Cells lacking endogenous T cell receptors and B2M do not exhibit graft versus host disease (GvHD) and are less susceptible to rejection by alloreactive host immune cells, but are vulnerable to NK cell lysis due to their lack of expression of HLA class I molecules. HLA-E and HLA-G proteins are minimally polymorphic, nonclassical HLA class I molecules that are ligands for NK cell inhibitory receptors. Their presence on the surface of cells therefore can reduce or prevent NK cell-mediated lysis, without triggering alloreactivity from host immune cells. Thus, in some aspects, the present disclosure provides modified cells comprising a TFP as provided herein, which have been engineered to knock down and/or knock out expression one or more endogenous TCR molecule and B2M, and which further comprise a peptide, protein, fusion protein, or other signal that reduces susceptibility to NK cell-mediated lysis. In some aspects, the peptide, protein, fusion protein, or signal is an agent that inhibits, reduces, prevents, or circumvents NK cell lysis and/or macrophage phagocytosis. Thus, provided herein are compositions of matter and methods of use for the treatment of a disease such as cancer, using modified T cells comprising a T cell receptor (TCR) fusion protein (TFP) in combination with a polypeptide or fusion protein that reduces, inhibits, eliminates, or prevents lysis of the modified T cells by NK cells. Exemplary polypeptides and fusion proteins include HLA- E and/or HLA-G, and/or fusions of B2M and/or HLA-E or HLA-G, including mutant B2M-HLA-E and/or mutant B2M-HLA-G. In some embodiments, the modified cells comprise mutB2M-HLA-E. In some embodiments, the modified cells comprise mutB2M-HLA-G. In some embodiments, the modified cells comprise both HLA-E and HLA-G polypeptides. For example, in some embodiments, the cells can comprise a TFP, a B2M-HLA-E fusion protein, and a B2M-HLA-G fusion protein. Advantageously, when the HLA-E and/or HLA-G polypeptides or fusion proteins are expressed in combination with the TFP in a cell which lacks HLA class I molecules (e.g., via knockout or knock down of B2M), they can confer resistance to NK cell mediated cytotoxicity, to which the cells would otherwise be susceptible due to the “missing self’ signal (absence of HLA class I). Thus, in some embodiments, expression of the peptide, protein, fusion protein, and/or signal provided herein on modified T cells provided herein confers increased persistence, prolonged activity, and increased efficacy on the T cells for treating the malignancies described herein. In some aspects, the modified cells provided herein are particularly useful for allogeneic or “off the shelf’ T cell therapies.

T cell receptor (TCR) fusion proteins (TFPs)

[0184] The present disclosure encompasses recombinant nucleic acid constructs encoding TFPs, wherein the

TFP comprises a binding domain, e.g., an antibody or antibody fragment, a ligand, or a ligand binding protein, wherein the sequence of the binding domain is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof. The binding domain can be an antibody domain comprising an antigen binding domain. The present disclosure encompasses recombinant nucleic acid constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to a tumor associated antigen (TAA) wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof. The present disclosure encompasses recombinant nucleic acid constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to a target (e.g., mesothelin), 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 encompasses recombinant nucleic acid constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to mesothelin, e.g., human mesothelin, 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 can 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.

[0185] 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).

[0186] 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.

[0187] 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.

[0188] Thus, in one aspect, the antigen-binding domain comprises a murine, 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-CD19 binding domain described herein, e.g. , a murine, humanized or human anti-TAA binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. In one embodiment, the murine, humanized or human anti-CD19 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., a scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity with an amino acid sequence provided herein; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein. In one embodiment, the 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. [0189] In some embodiments, the antigen-binding domain comprises an anti-mesothelin murine, 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 or 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. In some embodiments, the anti-mesothelin antibody has a variable domain of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71.

[0190] 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.

[0191] 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, 2Q( > .26^rl-^r19 (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. 5zo/., 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.)

[0192] A humanized antibody or antibody fragment has one or more amino acid residues remaining in it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. As provided herein, humanized antibodies or antibody fragments comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions wherein the amino acid residues comprising the framework are derived completely or mostly from human germline. Multiple techniques for humanization of antibodies or antibody fragments are well-known in the art and can essentially be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534- 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239,400; PCT Publication No. WO 91/09967; and U.S. 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.

[0193] 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. Immuno , 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.

[0194] 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.

[0195] 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 MSLN, or another tumor associated antigen.

[0196] In one aspect, the binding domain is characterized by particular functional features or properties of an antibody or antibody fragment. For example, in one aspect, the portion of a TFP composition of the present disclosure that comprises an antigen binding domain specifically binds human MSLN. In one aspect, the present disclosure relates to an antigen binding domain comprising an antibody or antibody fragment, wherein the antibody binding domain specifically binds to a MSLN protein or fragment thereof, wherein the antibody or antibody fragment comprises a variable light chain and/or a variable heavy chain that includes an amino acid sequence provided herein. In certain aspects, the scFv is contiguous with and in the same reading frame as a leader sequence.

[0197] In one aspect, the anti-tumor-associated antigen binding domain is a fragment, e.g., a single chain variable fragment (scFv). In one aspect, the anti-TAA binding domain is a Fv, a Fab, a (Fab’)2, or a bi- functional (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 CD 19 protein with wild-type or enhanced affinity. In another aspect, the anti-TAA binding domain comprises a single domain antibody (sdAb or VHH).

[0198] Also provided herein are methods for obtaining an antibody antigen binding domain specific for a target antigen (e.g., a 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 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/VL 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) and optionally with one or more desired properties.

[0199] 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. Set. USA 85:5879-5883). scFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The linker length can greatly affect how the variable regions of a scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5-10 amino acids) intra-chain folding is prevented. Inter-chain folding is also required to bring the two variable regions together to form a functional epitope binding site. In some instances, the linker sequence comprises a linker sequence. In some instances, the long linker sequence comprises (G4S)_n, wherein n=2 to 4. In some instances, the linker sequence comprises (G4S)_n, wherein n=l to 3. For examples of linker orientation and size see, e.g., Hollinger et al., 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT publication Nos. W02006/020258 and W02007/024715, each of which is incorporated herein by reference.

[0200] 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 embodiments, the linker sequence may comprise SEQ ID NO: 401.

Stability and Mutations

[0201] The stability of a 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.

[0202] The improved thermal stability of the anti-TAA 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 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 binding domain, has a 1 °C improved thermal stability as compared to a conventional antibody. In another embodiment, the binding domain, has a 2 °C improved thermal stability as compared to a conventional antibody. In another embodiment, the scFv has a 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, or 15 °C improved thermal stability as compared to a conventional antibody. Comparisons can be made, for example, between the scFv molecules disclosed herein and scFv molecules or Fab fragments of an antibody from which the scFv VH and VL were derived. Thermal stability can be measured using methods known in the art. For example, in one embodiment, TM can be measured. Methods for measuring TM and other methods of determining protein stability are described in more detail below. [0203] Mutations in antibody sequences (arising through humanization or direct mutagenesis of the soluble scFv) alter the stability of the antibody or fragment thereof and improve the overall stability of the antibody and the 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 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.

[0204] In one aspect, the antigen binding domain of the TFP comprises an amino acid sequence that is homologous to an antigen binding domain amino acid sequence described herein, and the antigen binding domain retains the desired functional properties of the anti-tumor-associated antigen antibody fragments described herein. In one specific aspect, the TFP composition of the present disclosure comprises an antibody fragment. In a further aspect, that antibody fragment comprises a scFv.

[0205] 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.

[0206] 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.

[0207] 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).

[0208] 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.

[0209] 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) ./. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Natl. 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.

[0210] In one aspect, the present disclosure contemplates modifications of the starting antibody or fragment (e.g., scFv) amino acid sequence that generate functionally equivalent molecules. For example, the VH or VL of a binding domain, e.g., scFv, comprised in the TFP can be modified to retain at least about 70%, 71%.

72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting VH or VL framework region of the anti-CD19 binding domain, e.g., scFv. 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%, 99% identity of the starting TFP construct.

Extracellular domain

[0211] 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 CD154. 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.

[0212] 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 an IgC domain 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 constant domain 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 constant domain but not the variable domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain. The extracellular domain can comprise a full-length extracellular 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.

[0213] 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.

[0214] 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 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 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 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.

[0215] 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 N- and C-terminus.

[0216] 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.

[0217] 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. [0218] 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

[0219] 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.

[0220] 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 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.

[0221] 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.

[0222] 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

[0223] 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 or a sequence (GGGGS)x or (G4S)_n, wherein X or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. In some embodiments, X or n is an integer from 1 to 10. In some embodiments, X or n is an integer from 1 to 4. In some embodiments, X or n is 2. In some embodiments, X or n is 4. In some embodiments, the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC. In some embodiments, the linker comprises a sequence of AAAGGGGSGGGGSGGGGSLE (SEQ ID NO:387).

Cytoplasmic Domain

[0224] 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, they are 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.

[0225] 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. 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. 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.

[0226] 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.

[0227] 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 antigenindependent manner to provide a secondary or costimulatory signal (secondary cytoplasmic domain, e.g. , a costimulatory domain). [0228] 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). [0229] 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. 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. [0230] 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, PD-1, 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).

[0231] 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., , 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences.

[0232] 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.

[0233] In one aspect, the TFPs described herein may comprise a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR alpha. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR beta. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR gamma. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR delta. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 epsilon. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 delta. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 gamma.

[0234] In one aspect, the TFPs described herein may comprise a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain, wherein all three of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from the same TCR subunit. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 epsilon. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 delta. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 gamma. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain may comprise the constant domain of TCR alpha. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain may comprise the constant domain of TCR beta. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain may comprise the constant domain of TCR gamma. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain may comprise the constant domain of TCR delta. In some embodiments, the constant domain of TCR alpha or the constant domain of TCR beta may be murine.

[0235] 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 a different target (e.g., CD70, 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.

IL-15 and IL-15 receptor alpha polypeptides

[0236] In some aspects, the TFP-expressing cells described herein can further express another agent such as an enhancing agent, for example, an agent that can enhance longevity or activity of TFP-expressing cells described herein. In some embodiments, the agent is a cytokine such as a pleiotropic cytokine that plays important roles in maintenance and homeostatic expansion of immune cells. In some embodiments, local secretion of a pleiotropic cytokine in tumor microenvironment (TME) can contribute to enhanced anti-tumor immunity. In some embodiments, the agent activates a cytokine signaling. In some embodiments the agent activates interleukin- 15 (IL-15) signaling. In some embodiments the agent comprises interleukin- 15 (IL-15) and/or interleukin- 15 receptor (IL-15R). In some embodiments, the IL-15R is an IL-15R alpha (IL-15Ra) subunit.

[0237] The present disclosure encompasses recombinant nucleic acid molecules encoding an interleukin- 15 (IL- 15) polypeptide or a fragment thereof. In some embodiments, the IL- 15 polypeptide or a fragment thereof comprises 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,

115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or more consecutive amino acid residues of IL-15. In some embodiments, the IL- 15 polypeptide or a fragment thereof 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 IL-15. In some embodiments, the IL- 15 polypeptide or a fragment thereof comprises a sequence encoding IL-15 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.

The recombinant nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof can be contained in the same nucleic acid molecule encoding the TFP described herein. The recombinant nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof can be contained in a nucleic acid molecule different from the nucleic acid sequence encoding the TFP described herein.

[0238] 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: 385. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 374. 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: 385. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 375. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise amino acids 1-162 of SEQ ID NO: 385. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 374 and a sequence of SEQ ID NO: 375. In some embodiments, the IL-15 polypeptide comprises a sequence of SEQ ID NO: 385. In some embodiments, IL- 15 polypeptide is secreted when expressed in a cell, such as a T cell.

[0239] The present disclosure further encompasses recombinant nucleic acid molecules encoding an interleukin- 15 receptor (IL-15R) subunit polypeptide or a fragment thereof. For example, the IL-15R subunit may be IL-15 receptor alpha chain (“IL-15Ra” or CD215), IL-2 receptor beta chain (“IL-2R[3” or CD122) and IL-2 receptor gamma/the common gamma chain (“IL-2Ry/yc” or CD132). In some embodiments, the IL-15R subunit is an IL-15Ra or a fragment thereof. In some embodiments, the IL-15Ra polypeptide or a fragment thereof comprises 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,

114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,

136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,

158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,

180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,

202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,

224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,

246, 247, 248, 249, 250, or more consecutive amino acid residues of IL-15Ra. In some embodiments, the IL- 15Ra polypeptide or a fragment thereof 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 IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof comprises a sequence encoding IL-15Ra 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, 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 amino acids at the N- or C- terminus or at both the N- and C-terminus.

[0240] 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: 386. 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: 386.

[0241] 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: 386. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 382.

[0242] 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: 386. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 372. [0243] 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: 386. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 382. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 383. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 96-267 of SEQ ID NO: 386. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 382 and a sequence of SEQ ID NO: 383. In some embodiments, IL- 15Ra comprises a sequence of SEQ ID NO: 403.

[0244] 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: 386. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 379.

[0245] 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 an integer from 1 to 4. In some embodiments, n is 3. In some embodiments, the linker comprises a sequence of SEQ ID NO: 378. In some embodiments, the linker comprises a sequence of SEQ ID NO: 405.

[0246] 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: 385. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO: 375. 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: 385. In some embodiments, the fusion protein does not comprise a sequence of SEQ ID NO: 374.

[0247] 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: 386. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO: 382. [0248] 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: 386. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO: 372.

[0249] 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: 386. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO: 379.

[0250] 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: 386. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO: 383. [0251] 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: 386. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO: 382 and a sequence of SEQ ID NO: 383. In some embodiments, IL-15Ra comprises a sequence of SEQ ID NO: 403.

[0252] In some embodiments, the fusion protein further comprises an epitope tag. An epitope tag as described herein can be a peptide epitope tag or a protein epitope tag. Examples of a peptide epitope tag includes, but are not limited to, 6X His (also known as His-tag or hexahistidine tag), FLAG (e.g., 3X FLAG), HA, Myc, and V5. Examples of a protein epitope tag include, but are not limited to, green fluorescent protein (GFP), glutathione-S-transferase (GST), [3-galactosidase ( -GAL), Luciferase, Maltose Binding Protein (MBP), Red Fluorescence Protein (RFP), and Vesicular Stomatitis Virus Glycoprotein (VSV-G). In some embodiments, the fusion protein further comprises a FLAG tag. In some embodiments, the fusion protein further comprises a 3X FLAG tag.

[0253] In some embodiments, the fusion protein is expressed on cell surface when expressed in a T cell. In some embodiments, the fusion protein is secreted when expressed in a T cell.

[0254] Disclosed herein, in some embodiments, are polypeptides encoded by any of recombinant nucleic acid molecules described herein.

HLA-E and HLA-G

[0255] HLA-E is a nonclassical MHC class lb antigen that is minimally polymorphic, with two functional alleles (HLA-E*01:01 and HLA-E*01:03), which differ by a single amino acid. HLA-E forms a heterodimer with a B2M subunit and typically presents peptides derived from leader sequences of other HLA class I molecules. HLA-E is the cognate ligand for the NK cell inhibitor receptor NKG2A; absence of HLA-E serves as a “missing self’ signal to NK cells that express NKG2A, and triggers NKG2A-mediated NK cell lysis. Fusion proteins of HLA-E-B2M or HLA-B2M and a peptide antigen (e.g., HLA-G binding peptide), when expressed on a cell surface, are able to confer resistance to NK cell-mediated lysis of cells that do not express other surface MHC molecules. HLA-G also has limited polymorphism and is associated with expression at the maternal -fetal interface in both membrane-bound and soluble forms, as a mechanism of maternal tolerance. Absence of HLA-G triggers NK cell activation via its receptors ILT2 or KIR2DL4. About 20-25% of NK cells in the peripheral blood are ILT2+ or KIR2DL4+, and about 50% of NK cells in the peripheral blood are NKG2A+. These inhibitory NK cell receptors have an immunoreceptor tyrosine-based inhibitory motif (ITIM) sequence which is phosphorylated upon ligand binding, and mediates the inhibition of NK cell activity.

[0256] An exemplary B2M-HLA-E fusion protein that includes HLA-G binding peptide is provided herein as SEQ ID NO: 423.

Recombinant Nucleic Acid Molecules

[0257] Disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) described herein. The recombinant nucleic acid molecule may comprise a second nucleic acid sequence encoding a TCR constant domain. The TCR constant domain can comprise (i) a TCR alpha constant domain, (ii) a TCR beta constant domain, (iii) a TCR alpha constant domain and a TCR beta constant domain, (iv) a TCR gamma constant domain, (v) a TCR delta constant domain, or (vi) a TCR gamma constant domain and a TCR delta constant domain. The recombinant nucleic acid molecule may comprise a third nucleic acid sequence encoding an Interleukin- 15 (IL- 15) polypeptide or a fragment thereof, and/or an NK cell inhibitor agent provided herein. In some cases, disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) and a second nucleic acid sequence encoding an Interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof. Also disclosed herein are recombinant nucleic acid molecules a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) and a second 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.

[0258] Disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) described herein; a second nucleic acid sequence encoding a TCR constant domain. The TCR constant domain can comprise (i) a TCR alpha constant domain, (ii) a TCR beta constant domain, (iii) a TCR alpha constant domain and a TCR beta constant domain, (iv) a TCR gamma constant domain, (v) a TCR delta constant domain, or (vi) a TCR gamma constant domain and a TCR delta constant domain; and a third nucleic acid sequence encoding an NK cell inhibitor agent provided herein. Disclosed herein, in some embodiments, are polypeptides encoded by any of recombinant nucleic acid molecules described herein.

Recombinant Nucleic Acid Encoding a TFP and a TCR Constant Domain [0259] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP). The TFP can comprise a TCR subunit comprising at least a portion of a TCR extracellular domain. The TCR subunit can further comprise a transmembrane domain. The TCR subunit can further comprise an intracellular domain of TCR gamma, TCR delta, TCR alpha or TCR beta or an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, CD3 delta. The TFP can further comprise an antibody (e.g., a human, humanized, or murine antibody) comprising an antigen binding domain. The recombinant nucleic acid molecule 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 antibody 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. Cells containing the TFPs described herein can be allogeneic cells. The recombinant nucleic acid molecules encoding the TCR constant domains can be used to prepare allogeneic cells for treating a subject in need thereof.

[0260] The sequence encoding the TFP and the sequence encoding the constant domain can be contained within the same recombinant nucleic acid molecule or two different recombinant nucleic acid molecules. [0261] 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.

[0262] 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.

[0263] 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: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:243 or SEQ ID NO:265. 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: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:243 or SEQ ID NO:265. 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: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:243 or SEQ ID NO:265. The constant domain can comprise a sequence or fragment thereof of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:243 or SEQ ID NO:265. 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: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 243 or SEQ ID NO: 265. 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: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:243 or SEQ ID NO:265. 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: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:243 or SEQ ID NO:265. In some cases, the TCR delta or the TCR delta constant domain comprises a sequence of SEQ ID NO: 243. In some cases, the TCR gamma or the TCR gamma constant domain comprises a sequence of SEQ ID NO: 21.

[0264] The murine TCR alpha constant domain can comprise positions 2-137 of SEQ ID NO: 146. 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 positions 2-137 of SEQ ID NO: 146. 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 positions 2-137 of SEQ ID NO: 146. 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 positions 2-137 of SEQ ID NO: 146. The constant domain can comprise a sequence or fragment thereof of positions 2-137 of SEQ ID NO: 146. 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 positions 2-137 of SEQ ID NO: 146. 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 positions 2-137 of SEQ ID NO: 146. 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 positions 2-137 of SEQ ID NO: 146.

[0265] The murine TCR beta constant domain can comprise positions 2-173 of SEQ ID NO: 152. 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 positions 2-173 of SEQ ID NO: 152. 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 positions 2-173 of SEQ ID NO: 152. 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 positions 2-173 of SEQ ID NO: 152. The constant domain can comprise a sequence or fragment thereof of positions 22-173 of SEQ ID NO: 152. 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 positions 2-173 of SEQ ID NO: 152. 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 positions 2-173 of SEQ ID NO: 152. 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 positions 2-173 of SEQ ID NO: 152.

[0266] In some instances, the TCR constant domain is a TCR delta constant domain. The TCR delta constant domain can comprise SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243 or SEQ ID NO:265, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modification. In some embodiments, the TCR delta constant domain can comprise SEQ ID NO:243. The TCR delta 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:243. 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:243. 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:243. The constant domain can comprise a sequence or fragment thereof of SEQ ID NO:243. 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:243. 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:243. 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:243.

[0267] The TCR delta constant domain can comprise SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243 or SEQ ID NO:265, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications. In some cases, the sequence encoding a TCR delta constant domain further encodes a TCR delta variable domain, thereby encoding a full TCR delta domain. The full TCR delta domain can be delta 2 or delta 1. The full TCR delta constant domain can comprise SEQ ID NO:256, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. [0268] The full TCR delta domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein. For example, the delta domain can comprise a truncated version of a delta 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:256. For example, the delta 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:256. For example, the delta 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:256. The delta domain can comprise a sequence or fragment thereof of SEQ ID NO:256. The delta 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:256. The delta 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:256. The delta 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:256.

[0269] The TCR gamma constant domain can comprise SEQ ID NO:21. The TCR gamma 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:21. 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:21. 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:21. The constant domain can comprise a sequence or fragment thereof of SEQ ID NO:21. 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:21. 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:21. 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:243. [0270] The TCR gamma constant domain can comprise SEQ ID NO:21 or SEQ ID NO: 155, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some cases, the sequence encoding the TCR gamma constant domain further encodes a TCR gamma variable domain, thereby encoding a full TCR gamma domain. The full TCR gamma domain can be gamma 9 or gamma 4. The full TCR gamma domain can comprise SEQ ID NO:255, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0271] The full TCR gamma domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein. For example, the gamma domain can comprise a truncated version of a gamma 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:255. For example, the gamma 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:255. For example, the gamma 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:255. The gamma domain can comprise a sequence or fragment thereof of SEQ ID NO:255. The gamma domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or gamma of the sequence of SEQ ID NO:255. The gamma 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:255. The gamma 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:255.

[0272] TCR beta chain (Homo sapiens): VEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQ PALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCG FTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 16).

[0273] The murine TCR beta chain constant region canonical sequence is: EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKES NYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSAS YQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 152).

[0274] TCR alpha constant region (Mus musculus) (or [mm]TRAC(82-137)): ATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 17).

[0275] The murine TCR alpha chain constant (mTRAC) region canonical sequence is:

XIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSN QTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRL WSS (SEQ ID NO: 146).

[0276] TCR beta constant region (Mus musculus) (or [mm]TRBCl(123-173)):

GRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 18).

[0277] The murine TCR beta chain constant region canonical sequence is:

EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKES NYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSAS YQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 152).

[0278] TCR beta chain (Homo sapiens):

PVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVSWYQQSLDQGLQFLIQYYNGEERAKGNILERFSA QQFPDLHSELNLSSLELGDSALYFCASSPRTGLNTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEI SHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFW QNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLG KATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 19).

[0279] TCR delta constant region version 1 (Homo sapiens):

SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSV TCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRML FAKTVAVNFLLTAKLFF (SEQ ID NO: 20).

[0280] TCR gamma constant region (Homo sapiens) (or [hs]TRGC( 1-173)):

DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDT YMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNT SAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS (SEQ ID NO: 21).

[0281] TCR delta constant region version 2 (Homo sapiens):

SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSV TCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRML FAKTVAVNFLLTAK (SEQ ID NO: 22).

[0282] In some instances, the TCR constant domain is a TCR delta constant domain. The sequence encoding the TCR delta constant domain can further encode a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR delta constant domain. The second antigen binding domain or ligand binding domain can be the same or different as the antigen binding domain or ligand binding domain of the TFP.

[0283] In some instances, the TCR constant domain is a TCR gamma constant domain. The sequence encoding the TCR gamma constant domain can further encode a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR gamma constant domain. The second antigen binding domain or ligand binding domain can be the same or different as the antigen binding domain or ligand binding domain of the TFP.

[0284] In some instances, the recombinant nucleic acid comprises a sequence encoding a TCR gamma constant domain and a TCR delta constant domain. The TCR gamma constant domain can comprise SEQ ID NO:21 or SEQ ID NO: 155, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. The sequence encoding the TCR gamma constant domain can further encode a TCR gamma variable domain, thereby encoding a full TCR gamma domain. The TCR gamma domain can be gamma 9 or gamma 4. The full TCR gamma domain comprises SEQ ID NO:255, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. The TCR delta constant domain can comprise SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243 or SEQ ID NO:265, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. The sequence encoding the TCR delta constant domain can further encode a TCR delta variable domain, thereby encoding a full TCR delta domain. The TCR delta domain can be delta 2 or delta 1. The full TCR delta domain can comprise SEQ ID NO:256, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0285] In some instances, the TCR constant domain incorporates into a functional TCR complex when expressed in a T cell. In some instances, the TCR constant domain incorporates into a same functional TCR complex as the functional TCR complex that incorporates the TFP when expressed in a T cell. In some instances, the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained within a same nucleic acid molecule. In some instances, the sequence encoding the TFP and the sequence encoding the TCR constant domain are 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. The cleavage site can comprise a sequence of SEQ ID NO: 23.

[0286] T2A cleavage site: EGRGSLLTCGDVEENPGP (SEQ ID NO: 23).

[0287] The TCR subunit of the TFP and the constant domain can comprise a sequence derived from a same TCR chain or a different TCR chain. In some cases, the TCR subunit of the TFP and the constant domain are derived from different TCR chains. For example, the TCR subunit can comprise (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, where the TCR extracellular domain, the transmembrane domain and the intracellular domain are derived from a TCR alpha chain, and the constant domain can comprise a constant domain of a TCR beta chain. For another example, the TCR subunit can comprise (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, where the TCR extracellular domain, the transmembrane domain and the intracellular domain are derived from a TCR beta chain, and the constant domain can comprise a constant domain of a TCR alpha chain. For another example, the TCR subunit can comprise (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, where the TCR extracellular domain, the transmembrane domain and the intracellular domain are derived from a TCR gamma chain, and the constant domain can comprise a constant domain of a TCR delta chain. For yet another example, the TCR subunit can comprise (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, where the TCR extracellular domain, the transmembrane domain and the intracellular domain are derived from a TCR delta chain, and the constant domain can comprise a constant domain of a TCR gamma chain.

[0288] In some instances, the TCR subunit and the antibody domain, the antigen domain or the binding ligand or fragment thereof are operatively linked by a linker sequence. In some instances, the linker sequence comprises (G4S)_n, wherein n=l to 4.

[0289] In some instances, the transmembrane domain is a TCR transmembrane domain from CD3 epsilon, CD3 gamma, CD3 delta, TCR gamma, TCR delta, TCR alpha or TCR beta. In some instances, the intracellular domain is derived from only CD3 epsilon, only CD3 gamma, only CD3 delta, only TCR gamma, only TCR delta, only TCR alpha or only TCR beta.

[0290] In some instances, the TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two or all of (i), (ii), and (iii) are from the same TCR subunit.

[0291] 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.

[0292] In some instances, the TCR 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 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. [0293] In some instances, the TCR subunit comprises a TCR intracellular domain of TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, or a fragment thereof. In some instances, the TCR subunit comprises an intracellular domain comprising a stimulatory domain of a protein 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.

[0294] In some instances, the TCR subunit can comprise (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain of a TCR gamma chain or a TCR delta chain. The TCR extracellular domain can comprise the extracellular portion of a constant domain of a TCR gamma chain or a TCR delta chain, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain is or comprises a delta constant domain, or a fragment thereof, e.g. , a delta constant domain described herein. The delta constant domain can have the sequence of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243 or SEQ ID NO:265, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain is or comprises a gamma constant domain, e.g., a gamma constant domain described herein. The gamma constant domain can have the sequence of SEQ ID NO:21 or SEQ ID NO: 155, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. The extracellular domain of the TFP may not comprise the variable domain of a gamma chain or a delta chain. [0295] The TCR subunit of the TFP can comprise the extracellular, transmembrane and intracellular domain of CD3 epsilon, CD3 gamma, or CD3 delta. In some embodiments, recombinant nucleic acid comprises a TFP comprising the extracellular, transmembrane and intracellular domain of CD3 epsilon, CD3 gamma, or CD3 delta and the constant domains of TCR beta and TCR alpha. In some embodiments, recombinant nucleic acid comprises a TFP comprising the extracellular, transmembrane and intracellular domain of CD3 epsilon and the constant domains of TCR gamma and TCR delta. In some embodiments, recombinant nucleic acid comprises a TFP comprising the extracellular, transmembrane and intracellular domain of CD3 epsilon and full length TCF gamma and full length TCR delta. In some embodiments, the TCR subunit of the TFP comprises CD3 epsilon. The TCR subunit of CD3 epsilon can comprise the sequence of SEQ ID NO:258 functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0296] In some instances, the TCR subunit comprising at least a portion of a murine TCR alpha or murine TCR beta extracellular domain and a murine TCR alpha or murine TCR beta transmembrane domain is or comprises a TCR alpha constant domain or a TCR beta constant domain. The TCR subunit can comprise an intracellular domain of murine TCR alpha or murine TCR beta. The TCR constant domain can be a TCR alpha constant domain, e.g., a TCR alpha constant domain described herein. The TCR alpha constant domain can comprise SEQ ID NO: 17, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO: 207, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. The sequence encoding the TCR alpha constant domain can further encode a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR alpha constant domain. The second antigen binding domain or ligand binding domain can be the same or different as the antigen binding domain or ligand binding domain of the TFP. The TCR alpha constant domain can comprise a murine TCR alpha constant domain. The murine TCR alpha constant domain can comprise amino acids 2-137 of the murine TCR alpha constant domain. The murine TCR alpha constant domain can comprise amino acids 2-137 of SEQ ID NO: 146. The murine TCR alpha constant domain can comprise a sequence of SEQ ID NO:207. The murine TCR alpha constant domain can comprise amino acids 82-137 of SEQ ID NO: 146. The murine TCR alpha constant domain comprises a sequence of SEQ ID NO: 17. The TCR constant domain can be a TCR beta constant domain, e.g., a TCR beta constant domain described herein. The TCR beta constant domain can comprise SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO:209, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. The sequence encoding the TCR beta constant domain can further encode a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR beta constant domain. The second antigen binding domain or ligand binding domain can be the same or different as the antigen binding domain or ligand binding domain of the TFP. TCR beta constant domain can comprise a murine TCR beta constant domain. The murine TCR beta constant domain can comprise amino acids 2-173 of the murine TCR beta constant domain. The murine TCR beta constant domain can comprise amino acids 2-173 of SEQ ID NO: 152. The murine TCR beta constant domain can comprise SEQ ID NO:209. The TCR beta constant domain can comprise amino acids 123-173 of SEQ ID NO: 152. The TCR beta constant domain can comprise SEQ ID NO: 18

[0297] The recombinant nucleic acid can comprise sequence encoding a TCR alpha constant domain and a TCR beta constant domain. The TCR alpha constant domain can comprise SEQ ID NO: 17, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO:207, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. The TCR beta constant domain can comprise SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO:209, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. The intracellular signaling domain can be CD3 epsilon, CD3 gamma, or CD3 delta. The intracellular signaling domain can be CD3 epsilon.

[0298] The sequence encoding the TCR constant domain can comprise from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene sequence. The sequence encoding the TCR constant domain can comprise, from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene sequence. The sequence encoding the TCR constant domain can comprise, from 5’ to 3’, a first leader sequence, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker, and a TRBC gene sequence. The sequence encoding the TCR constant domain can comprise, from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker, and a TRBC gene sequence. The sequence encoding the TCR constant domain can comprise, from 5 ’-3’, a first leader sequence, a TRAC gene sequence, a first cleavable linker sequence, a second leader sequence, a TRBC gene sequence, a second cleavable linker sequence, a third leader sequence, an antigen binding domain sequence, a linker sequence, and a CD 3 epsilon gene sequence.

[0299] As described herein, the at least one but not more than 20 modifications thereto of a sequence described herein can comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP. [0300] In some instances, the TCR subunit comprises an intracellular domain comprising a stimulatory domain of a protein selected from a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.

[0301] In some instances, the recombinant nucleic acid further comprises a sequence encoding a costimulatory domain. In some instances, the costimulatory domain comprises a functional signaling domain of 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), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.

[0302] In some instances, the TCR subunit comprises 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 (DAP 12), CD5, CD 16a, CD 16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto. In some instances, the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon. In some instances, the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.

[0303] In some instances, the TFP, the TCR gamma constant domain, the TCR delta constant domain, and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide. In some instances, (a) the TCR constant domain is a TCR gamma constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR delta, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof; (b) the TCR constant domain is a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR gamma, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof; or (c) the TCR constant domain is a TCR gamma constant domain and a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof.

[0304] In some instances, the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.

[0305] The antibody or antigen binding domain can be an antibody fragment. The antibody or antigen binding domain can be murine, human or humanized. In some instances, the murine, human or humanized antibody is an antibody fragment. In some instances, the antibody fragment is a scFv, a single domain antibody domain, a VH domain or a VL domain. In some instances, murine, human or humanized antibody comprising an antigen binding domain is selected from a group consisting of an anti-CD19 binding domain, anti-B-cell maturation antigen (BCMA) binding domain, anti-mesothelin (MSLN) binding domain, anti-CD22 binding domain, anti-PD-1 binding domain, anti-BAFF or BAFF receptor binding domain, and anti-ROR-1 binding domain.

[0306] An antigen binding domain described herein can be selected from a group consisting of an anti-CD19 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, an anti-79b binding domain, an anti-HER2 binding domain, an anti-PMSA binding domain, an anti-MUC16 binding domain, an anti-CD22 binding domain, an anti-PD-Ll binding domain, an anti BAFF or BAFF receptor binding domain, an anti- Nectin-4 binding domain, an anti-TROP-2 binding domain, an anti-GPC3 binding domain, and anti-ROR-1 binding domain.

[0307] In some instances, the nucleic acid is selected from the group consisting of a DNA and an RNA. In some instances, the nucleic acid is an mRNA. In some instances, the recombinant nucleic acid comprises a nucleic acid analog, wherein the nucleic acid analog is not in an encoding sequence of the recombinant nucleic acid. In some instances, the nucleic analog is selected from the group consisting of 2’-O-methyl, 2’-O- methoxyethyl (2’-O-MOE), 2’-O-aminopropyl, 2’-deoxy, 2 ’-deoxy-2’ -fluoro, 2’-O-aminopropyl (2’-O-AP), 2'-O-dimethylaminoethyl (2’-O-DMAOE), 2’-O-dimethylaminopropyl (2’-O-DMAP), 2 -O- dimethylaminoethyloxyethyl (2’-O-DMAEOE), 2’-O-N-methylacetamido (2’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2’- fluoro N3-P5’-phosphoramidite.

[0308] 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. In some instances, the nucleic acid is an in vitro transcribed nucleic acid.

[0309] In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR beta transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain and a sequence encoding a TCR beta transmembrane domain.

[0310] In some instances, the TCR subunit comprises 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 (DAP 12), CD5, CD 16a, CD 16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto. In some instances, the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon. In some instances, the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.

[0311] In some instances, the TFP, the TCR gamma constant domain, the TCR delta constant domain, the TCR alpha constant domain, the TCR beta constant domain, and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide. In some instances, (a) the TCR constant domain is a TCR gamma constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR beta, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof; (b) the TCR constant domain is a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR alpha, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof; (c) the TCR constant domain is a TCR gamma constant domain and a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof; (d) the TCR constant domain is a TCR alpha constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR beta, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof; or (e) the TCR constant domain is a TCR beta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR alpha, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof.

[0312] In some instances, the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.

[0313] In some instances, the murine, human or humanized antibody is an antibody fragment. In some instances, the antibody fragment is a scFv, a single domain antibody domain (sdAb), a VH domain or a VL domain. In some instances, murine, human or humanized antibody comprising an antigen binding domain is selected from a group consisting of an anti-CD19 binding domain, anti-B-cell maturation antigen (BCMA) binding domain, anti-mesothelin (MSLN) binding domain, anti-CD22 binding domain, anti-PD-1 binding domain, anti PD-L1 binding domain, anti IL13Ra2 binding domain, anti-BAFF or BAFFR binding domain, and anti-ROR-1 binding domain.

[0314] In some instances, the TCR subunit and the antibody domain, the antigen domain or the binding ligand or fragment thereof are operatively linked by a linker sequence. In some instances, the linker sequence comprises (G4S)_n, wherein n=l to 4. [0315] In some instances, the transmembrane domain is a TCR transmembrane domain from CD3 epsilon, CD3 gamma, CD3 delta, TCR alpha, TCR beta, TCR delta, or TCR gamma. In some instances, the intracellular domain is derived from only CD3 epsilon, only CD3 gamma, only CD3 delta, only TCR alpha, only TCR beta, only TCR delta, or only TCR gamma.

[0316] In some instances, the TCR subunit comprises (i) at least a portion of 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.

[0317] 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 delta chain, a TCR gamma 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.

[0318] In some instances, the TCR 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 delta chain, a TCR gamma 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.

[0319] In some instances, the TFP, the TCR gamma constant domain, the TCR delta constant domain, the TCR alpha constant domain, the TCR beta constant domain, and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide. In some instances, (a) the TCR constant domain is a TCR gamma constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR beta, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof; (b) the TCR constant domain is a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR gamma, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof; (c) the TCR constant domain is a TCR gamma constant domain and a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof; (d) the TCR constant domain is a TCR alpha constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR beta, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof; or (e) the TCR constant domain is a TCR beta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR alpha, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof.

[0320] In some instances, the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP. [0321] In some instances, the murine, human or humanized antibody is an antibody fragment. In some instances, the antibody fragment is a scFv, a single domain antibody domain, a VH domain or a VL domain. In some instances, murine, human or humanized antibody comprising an antigen binding domain is selected from a group consisting of an anti-CD19 binding domain, anti-CD20 binding domain, anti-mesothelin binding domain, anti-PMSA binding domain, anti-CD70 binding domain, anti-CD79b binding domain, anti-MUC16 binding domain, anti-anti-B-cell maturation antigen (BCMA) binding domain, anti-mesothelin (MSLN) binding domain, anti-IL13Ra2 binding domain, anti-CD22 binding domain, anti-BAFF or anti-BAFFR binding domain, anti-PD-1 binding domain, anti-PD-Ll binding domain, and anti-ROR-1 binding domain. [0322] In some embodiments, a sequence encoding the antigen binding domain is operatively linked to a sequence encoding a delta constant domain. In some embodiments, the intracellular domain is an intracellular domain of TCR gamma. In some embodiments, a sequence encoding the antigen binding domain is operatively linked to a sequence encoding a gamma constant domain. In some embodiments, the intracellular domain is an intracellular domain of TCR delta. In some embodiments, a sequence encoding the antigen binding domain is operatively linked to both a sequence encoding a TCR delta constant domain or fragment thereof and a TCR gamma constant domain or fragment thereof. In some embodiments, the intracellular signaling domain is CD3 epsilon, CD3 gamma, or CD3 delta. In some embodiments, the intracellular signaling domain is CD3 epsilon. In some embodiments, the recombinant nucleic acid further comprises at least one leader sequence and at least one linker. In some embodiments, the recombinant nucleic acid further comprises a portion of a TCR alpha constant domain, a portion of a TCR beta domain, or both.

Recombinant Nucleic Acid Encoding IL-15 and/or IL-15Ra

[0323] Disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein and a second nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof. Any recombinant nucleic acid molecules comprising a nucleic acid sequence encoding a TFP described herein may further comprise a second nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof. Further disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein and a second nucleic acid sequence encoding an IL-15Ra polypeptide or a fragment thereof. Any recombinant nucleic acid molecules comprising a nucleic acid sequence encoding a TFP described herein may further comprise a second nucleic acid sequence encoding an IL-15Ra polypeptide or a fragment thereof.

[0324] Also disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein, a second nucleic acid sequence encoding a TCR constant domain described herein, and a third nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof. Also disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein, a second nucleic acid sequence encoding a TCR constant domain described herein, and a third nucleic acid sequence encoding an IL-15Ra polypeptide or a fragment thereof. Also disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein, a second nucleic acid sequence encoding a TCR constant domain described herein, and a third nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof and an IL-15Ra polypeptide or a fragment thereof (e.g., encoding a fusion protein of IL- 15 and IL-15Ra). The sequence encoding the TFP, the sequence encoding the TCR constant domain, and the sequence encoding an IL- 15 polypeptide (or a fragment thereof) or an IL-15Ra polypeptide (or a fragment thereof) can be contained in the same or different nucleic acid molecules. For example, the sequence encoding the TFP, the sequence encoding the TCR constant domain, and the sequence encoding an IL- 15 polypeptide (or a fragment thereof) or an IL-15Ra polypeptide (or a fragment thereof) can be contained in the same nucleic acid molecule. For another example, at least two of the sequences including the sequence encoding the TFP, the sequence encoding the TCR constant domain, and the sequence encoding an IL- 15 polypeptide (or a fragment thereof) or an IL-15Ra polypeptide (or a fragment thereof) can be contained in the same nucleic acid molecule. For another example, one of the sequences including the sequence encoding the TFP, the sequence encoding the TCR constant domain, and the sequence encoding an IL-15 polypeptide (or a fragment thereof) or an IL-15Ra polypeptide (or a fragment thereof) can be contained in a separate nucleic acid molecule from the other two sequences.

[0325] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein and a second nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof, wherein the first nucleic acid sequence and the second nucleic acid sequence are included in two separate nucleic acid molecules. Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein and a second nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof, wherein the first nucleic acid sequence and the second nucleic acid sequence are included in a single nucleic acid molecule. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a first linker. Further disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein and a second nucleic acid sequence encoding an IL-15Ra polypeptide or a fragment thereof, wherein the first nucleic acid sequence and the second nucleic acid sequence are included in two separate nucleic acid molecules. Further disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein and a second nucleic acid sequence encoding an IL-15Ra polypeptide or a fragment thereof, wherein the first nucleic acid sequence and the second nucleic acid sequence are included in a single nucleic acid molecule. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a first linker. For example, the first linker may be a cleavable linker. In some embodiments, the first 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 cleavage site can comprise a sequence of SEQ ID NO: 23, when expressed. In some embodiments, the first linker comprises a sequence of SEQ ID NO: 23, when expressed.

[0326] In some embodiments, the nucleic acid sequence encoding the IL- 15 polypeptide, or a fragment thereof may comprise a sequence encoding IL- 15 signal peptide. In some embodiments, IL- 15 signal peptide comprises amino acids 1-29 of SEQ ID NO: 385, when expressed. In some embodiments, IL-15 signal peptide comprises a sequence of SEQ ID NO: 374, when expressed. In some embodiments, the nucleic acid sequence encoding the IL- 15 polypeptide, or a fragment thereof may comprise a sequence encoding amino acids 30-162 of SEQ ID NO: 385. In some embodiments, the nucleic acid sequence encoding the IL-15 polypeptide, or a fragment thereof may comprise a sequence encoding a sequence of SEQ ID NO: 375. In some embodiments, the nucleic acid sequence encoding the IL- 15 polypeptide, or a fragment thereof may comprise a sequence encoding amino acids 1-162 of SEQ ID NO: 385. In some embodiments, the nucleic acid sequence encoding the IL-15 polypeptide, or a fragment thereof may comprise a sequence encoding a sequence of SEQ ID NO: 374 and a sequence of SEQ ID NO: 375. In some embodiments, the IL-15 polypeptide or a fragment thereof is secreted when expressed in a T cell. In some embodiments, the IL- 15 polypeptide comprises a sequence of SEQ ID NO: 375, when expressed.

[0327] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein and a second nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof and an IL-15R subunit or a fragment thereof, wherein the first nucleic acid sequence and the second nucleic acid sequence are included in two separate nucleic acid molecules. Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein and a second nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof and an IL-15R subunit or a fragment thereof, wherein the first nucleic acid sequence and the second nucleic acid sequence are included in a single nucleic acid molecule. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a first linker described herein. An IL-15R subunit may be an IL-15R alpha (IL-15Ra), an IL-2R beta (IL-2[3), or an IL-2R gamma/the common gamma chain (IL-2Ry/yc). In some embodiments, the IL-15R subunit is IL-15R alpha (IL-15Ra). In some embodiments, IL- 15 and IL-15R subunit are operatively linked by a second linker. In some embodiments, IL- 15 and IL-15Ra are operatively linked by a second linker. In some embodiments, the second linker is not a cleavable linker. For example, the second 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 an integer from 1 to 4. In some embodiments, n is 3. In some embodiments, the second linker comprises a sequence of SEQ ID NO: 378. In some embodiments, the second linker comprises a sequence of SEQ ID NO: 405.

[0328] In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding the intracellular domain of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding amino acids 229-267 of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the IL- 15Ra polypeptide or a fragment thereof may comprise a sequence encoding amino acids 229-267 of SEQ ID NO: 386. In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding a sequence of SEQ ID NO: 372. [0329] In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding IL-15Ra Sushi domain. In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding amino acids 31-95 of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the IL- 15 Ra polypeptide or a fragment thereof may comprise a sequence encoding amino acids 31-95 of SEQ ID NO: 386. In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding a sequence of SEQ ID NO: 382.

[0330] In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding the transmembrane domain and the intracellular domain of IL- 15Ra. In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding amino acids 96-267 of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding amino acids 96-267 of SEQ ID NO: 386. In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding a sequence of SEQ ID NO: 383.

[0331] In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding the Sushi domain, the transmembrane domain, and the intracellular domain of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding amino acids 31-267 of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding amino acids 31-267 of SEQ ID NO: 386. In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding a sequence of SEQ ID NO: 382 and a sequence of SEQ ID NO: 383. In some embodiments, IL-15Ra comprises a sequence of SEQ ID NO: 403.

[0332] In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding a soluble IL-15Ra (sIL-15Ra). In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding amino acids 21-205 of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding amino acids 21-205 of SEQ ID NO: 386. In some embodiments, the nucleic acid sequence encoding the IL-15Ra polypeptide or a fragment thereof may comprise a sequence encoding a sequence of SEQ ID NO: 379.

[0333] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein and a second nucleic acid sequence encoding a fusion protein comprising an IL- 15 polypeptide linked to an IL-15Ra subunit, wherein the first nucleic acid sequence and the second nucleic acid sequence are included in two separate nucleic acid molecules. Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein and a second nucleic acid sequence encoding a fusion protein comprising an IL- 15 polypeptide linked to an IL-15Ra subunit, wherein the first nucleic acid sequence and the second nucleic acid sequence are included in a single nucleic acid molecule. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a first linker described herein. 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.

[0334] In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 1-29 of IL- 15. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 1-29 of SEQ ID NO: 385. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 374. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 30-162 of IL-15. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 30-162 of SEQ ID NO: 385. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 375. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 1-162 of IL-15. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 1-162 of SEQ ID NO: 385. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 374 and a sequence encoding a sequence of SEQ ID NO: 375.

[0335] In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding the intracellular domain of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 229-267 of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 229-267 of SEQ ID NO: 386. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 372.

[0336] In some embodiments, the nucleic acid sequence encoding the fusion protein may further comprise a sequence encoding IL-15Ra Sushi domain. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 31-95 of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 31-95 of SEQ ID NO: 386. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 382.

[0337] In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding the transmembrane domain and the intracellular domain of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 96-267 of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 96-267 of SEQ ID NO: 386. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 383.

[0338] In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding the Sushi domain, the transmembrane domain, and the intracellular domain of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 31-267 of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 31-267 of SEQ ID NO: 386. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 382 and a sequence of SEQ ID NO: 383. In some embodiments, IL-15Ra comprises a sequence of SEQ ID NO: 403.

[0339] In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a soluble IL-15Ra (sIL-15Ra). In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 21-205 of IL-15Ra. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding amino acids 21-205 of SEQ ID NO: 386. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 379.

[0340] In some embodiments, the nucleic acid sequence encoding the fusion protein may further comprise a sequence encoding an epitope tag. An epitope tag as described herein can be a peptide epitope tag or a protein epitope tag. Examples of a peptide epitope tag includes, but are not limited to, 6X His (also known as His-tag or hexahistidine tag), FLAG (e.g. , 3X FLAG), HA, Myc, and V5. Examples of a protein epitope tag include, but are not limited to, green fluorescent protein (GFP), glutathione-S-transferase (GST), [3-galactosidase ([3- GAL), Luciferase, Maltose Binding Protein (MBP), Red Fluorescence Protein (RFP), and Vesicular Stomatitis Virus Glycoprotein (VSV-G). In some embodiments, the nucleic acid sequence encoding the fusion protein further comprises a sequence encoding a FLAG tag. In some embodiments, the nucleic acid sequence encoding the fusion protein further comprises a sequence encoding a 3X FLAG tag.

[0341] In some embodiments, the fusion protein is expressed on cell surface when expressed from the recombinant nucleic acid molecule described herein in a T cell. In some embodiments, the fusion protein is secreted when expressed from the recombinant nucleic acid molecule described herein in a T cell.

[0342] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein, a second nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof, and a third nucleic acid sequence encoding an agent that can enhance the activity of a modified T cell expressing the TFP. In some embodiments, the third nucleic acid sequence is included in a separate nucleic acid sequence. In some embodiments, the third nucleic acid sequence is included in the same nucleic acid molecule as the first nucleic acid sequence or the second nucleic acid sequence, or the first and the second nucleic acid sequences.

[0343] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein and a second nucleic acid sequence encoding an IL- 15Ra polypeptide or a fragment thereof, wherein the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a first linker described herein, and wherein the second nucleic acid sequence further encodes an agent that can enhance the activity of a modified T cell expressing the TFP. For example, the agent can be an agent that can inhibit an inhibitory molecule that can decrease the ability of a T cell expressing a TFP to mount an immune effector response.

[0344] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein, a second nucleic acid sequence encoding an IL-15Ra polypeptide or a fragment thereof and an agent that can enhance the activity of a modified T cell expressing the TFP described herein, and a third nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are included in two separate nucleic acid sequences. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are included in a single nucleic acid sequence. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a first linker described herein. In some embodiments, the third nucleic acid sequence is included in a separate nucleic acid molecule from the first nucleic acid sequence or the second nucleic acid sequence, or the first and the second nucleic acid sequences. In some embodiments, the third nucleic acid sequence is included in the same nucleic acid molecule as the first nucleic acid sequence or the second nucleic acid sequence, or the first and the second nucleic acid sequences. In some embodiments, the third nucleic acid sequence encoding the IL-15 polypeptide may comprise a sequence encoding amino acids 1-29 of IL-15. In some embodiments, the third nucleic acid sequence encoding the IL- 15 polypeptide may comprise a sequence encoding amino acids 1-29 of SEQ ID NO: 385. In some embodiments, the third nucleic acid sequence encoding the IL-15 polypeptide may comprise a sequence encoding a sequence of SEQ ID NO: 374. In some embodiments, the third nucleic acid sequence encoding the IL- 15 polypeptide may comprise a sequence encoding amino acids 30-162 of IL-15. In some embodiments, the third nucleic acid sequence encoding the IL- 15 polypeptide may comprise a sequence encoding amino acids 30-162 of SEQ ID NO: 385. In some embodiments, the third nucleic acid sequence encoding the IL-15 polypeptide may comprise a sequence encoding a sequence of SEQ ID NO: 375. In some embodiments, the IL-15 polypeptide is secreted when expressed in a T cell. In some embodiments, the third nucleic acid sequence encoding the IL- 15 polypeptide may comprise a sequence encoding amino acids 1-162 of IL-15. In some embodiments, the third nucleic acid sequence encoding the IL- 15 polypeptide may comprise a sequence encoding amino acids 1-162 of SEQ ID NO: 385. In some embodiments, the third nucleic acid sequence encoding the IL-15 polypeptide may comprise a sequence encoding a sequence of SEQ ID NO: 374 and a sequence of SEQ ID NO: 375.

[0345] The recombinant nucleic acid molecules can comprise a sequence of any of the nucleic acid sequences listed in Table 5. In some embodiments, the recombinant nucleic acid molecules can encode an amino acid sequence of any of the amino acid sequences listed in Table 5. For example, the recombinant nucleic acid can comprise a sequence encoding a signal peptide. The signal peptide can be a GM-CSF signal peptide. The recombinant nucleic acid molecule can further comprise a sequence encoding a protease. The protease can be a furin. The recombinant nucleic acid can comprise a sequence of SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 400, SEQ ID NO: 402, and/or SEQ ID NO: 404. The recombinant nucleic acid molecule can comprise a sequence encoding SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 69, SEQ ID NO: 243, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, and/or SEQ ID NO: 21. The recombinant nucleic acid molecule can encode, from N-terminus to C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a linker (e.g., GSG linker), operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional anti-MSLN antigen binding domain, operatively linked to a TCR gamma constant domain.

[0346] The recombinant nucleic acid can comprise a sequence of SEQ ID NO: 407, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 404, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, and/or SEQ ID NO: 411. The recombinant nucleic acid molecule can comprise a sequence encoding SEQ ID NO: 366, SEQ ID NO: 362, SEQ ID NO: 69, SEQ ID NO: 243, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 385, SEQ ID NO: 405, and/or SEQ ID NO: 403. The recombinant nucleic acid molecule can encode, from N-terminus to C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a linker (e.g., GSG linker), operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional anti- MSLN antigen binding domain, operatively linked to a TCR gamma constant domain, operatively linked to a linker, operatively linked to a T2A sequence, operatively linked to a IL- 15 polypeptide, operatively linker to a linker, operatively linked to a hIL-15Ra polypeptide.

[0347] The recombinant nucleic acid can comprise a sequence of SEQ ID NO: 412, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 413, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 414, and/or SEQ ID NO: 404. The recombinant nucleic acid molecule can encode a sequence of SEQ ID NO: 367, SEQ ID NO: 362, SEQ ID NO: 69, SEQ ID NO: 387, SEQ ID NO: 243, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, and/or SEQ ID NO: 21. The recombinant nucleic acid molecule can encode, from N-terminus to C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN antigen binding domain, operatively linked to a first linker, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a second linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional anti-MSLN antigen binding domain, operatively linked to a third linker, operatively linked to a TCR gamma constant domain.

[0348] The recombinant nucleic acid can comprise a sequence of SEQ ID NO: 415, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 413, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 414, SEQ ID NO: 404, SEQ ID NO: 390, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, and/or SEQ ID NO: 411. The recombinant nucleic acid molecule can encode a sequence of SEQ ID NO: 368, SEQ ID NO: 362, SEQ ID NO: 69, SEQ ID NO: 387, SEQ ID NO: 243, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 385, SEQ ID NO: 405, and/or SEQ ID NO: 403. The recombinant nucleic acid molecule can encode, from N-terminus to C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN antigen binding domain, operatively linked to a first linker, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a second linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional anti-MSLN antigen binding domain, operatively linked to a third linker, operatively linked to a TCR gamma constant domain, operatively linked to a fourth linker, operatively linked to a T2A sequence, operatively linked to a IL- 15 polypeptide, operatively linker to a linker, operatively linked to a hIL-15Ra polypeptide.

[0349] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a nucleic acid sequence encoding a TFP described herein, a nucleic acid sequence encoding an IL- 15 polypeptide, or a fragment thereof described herein, and a nucleic acid sequence encoding an IL-15Ra or a fragment thereof described herein. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a sequence encoding CSF2RA signal peptide. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 362. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a sequence encoding anti-MSLN antibody. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 69. In some embodiments, the nucleic acid sequence encoding IL-15 polypeptide or fragment thereof comprise a sequence encoding amino acids 1-29 of IL-15. In some embodiments, the nucleic acid sequence encoding IL- 15 polypeptide or fragment thereof comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 374. In some embodiments, the nucleic acid sequence encoding IL-15 polypeptide or fragment thereof may comprise a sequence encoding amino acids 30-162 of IL-15. In some embodiments, the nucleic acid sequence encoding IL- 15 polypeptide or fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 375. In some embodiments, the nucleic acid sequence encoding IL-15Ra polypeptide or fragment thereof comprise a sequence encoding amino acids 21-205 of IL- 15Ra. In some embodiments, the nucleic acid sequence encoding IL-15Ra polypeptide or fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 379. In some embodiments, the nucleic acid sequence encoding the TFP and the nucleic acid sequence encoding the IL- 15 polypeptide, or a fragment thereof are operatively linked by a T2A linker. In some embodiments, the T2A linker may comprise a sequence of SEQ ID NO: 23. In some embodiments, the nucleic acid sequence encoding the IL-15 polypeptide or a fragment thereof and the nucleic acid sequence encoding the IL-15Ra or a fragment thereof are operatively linked by a non-cleavable linker. In some embodiments, the non-cleavable linker may comprise a sequence of SEQ ID NO: 378. In some embodiments, the non-cleavable linker comprises a sequence of SEQ ID NO: 405. [0350] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a nucleic acid sequence encoding a TFP described herein and a nucleic acid sequence encoding an IL- 15 polypeptide, or a fragment thereof described herein. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a sequence encoding CSF2RA signal peptide. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 362. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a sequence encoding anti-MSLN antibody. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 69. In some embodiments, the nucleic acid sequence encoding IL- 15 polypeptide or fragment thereof comprise a sequence encoding amino acids 1-29 of IL-15. In some embodiments, the nucleic acid sequence encoding IL- 15 polypeptide or fragment thereof comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 374. In some embodiments, the nucleic acid sequence encoding IL- 15 polypeptide or fragment thereof may comprise a sequence encoding amino acids 30-162 of IL-15. In some embodiments, the nucleic acid sequence encoding IL- 15 polypeptide or fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 375. In some embodiments, the nucleic acid sequence encoding the TFP and the nucleic acid sequence encoding the IL- 15 polypeptide, or a fragment thereof are operatively linked by a T2A linker. In some embodiments, the T2A linker may comprise a sequence of SEQ ID NO: 23.

[0351] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a nucleic acid sequence encoding a TFP described herein, a nucleic acid sequence encoding an IL- 15 polypeptide, or a fragment thereof described herein, and a nucleic acid sequence encoding an IL-15Ra or a fragment thereof described herein. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a sequence encoding CSF2RA signal peptide. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 362. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a sequence encoding anti-MSLN antibody. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 69. In some embodiments, the nucleic acid sequence encoding IL-15 polypeptide or fragment thereof comprise a sequence encoding amino acids 1-29 of IL-15. In some embodiments, the nucleic acid sequence encoding IL- 15 polypeptide or fragment thereof comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 374. In some embodiments, the nucleic acid sequence encoding IL-15 polypeptide or fragment thereof may comprise a sequence encoding amino acids 30-162 of IL-15. In some embodiments, the nucleic acid sequence encoding IL- 15 polypeptide or fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 375. In some embodiments, the nucleic acid sequence encoding IL-15Ra polypeptide or fragment thereof comprise a sequence encoding amino acids 31-95 of IL- 15Ra. In some embodiments, the nucleic acid sequence encoding IL-15Ra polypeptide or fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 382. In some embodiments, the nucleic acid sequence encoding IL-15Ra polypeptide or fragment thereof comprise a sequence encoding amino acids 96-267 of IL-15Ra. In some embodiments, the nucleic acid sequence encoding IL-15Ra polypeptide or fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 383. In some embodiments, the nucleic acid sequence encoding IL-15Ra polypeptide or fragment thereof may comprise a nucleic acid sequence encoding SEQ ID NO: 403. In some embodiments, the nucleic acid sequence encoding the TFP and the nucleic acid sequence encoding the IL- 15 polypeptide, or a fragment thereof are operatively linked by a T2A linker. In some embodiments, the T2A linker may comprise a sequence of SEQ ID NO: 23. In some embodiments, the nucleic acid sequence encoding the IL-15 polypeptide or a fragment thereof and the nucleic acid sequence encoding the IL-15Ra or a fragment thereof are operatively linked by a non-cleavable linker. In some embodiments, the non-cleavable linker may comprise a sequence of SEQ ID NO: 378. In some embodiments, the non-cleavable linker may comprise a sequence of SEQ ID NO: 405. In some embodiments, the recombinant nucleic acid molecule may further comprise a sequence encoding a 3X FLAG tag.

[0352] In some embodiments, recombinant nucleic acid molecules described herein further comprise a leader sequence. In some embodiments, the recombinant nucleic acid molecule is selected from the group consisting of a DNA and an RNA. In some embodiments, the recombinant nucleic acid molecule is an mRNA. In some embodiments, the recombinant nucleic acid molecule is a circRNA. In some embodiments, the recombinant nucleic acid molecule comprises a nucleic acid analog. In some embodiments, the nucleic acid analog is not in an encoding sequence of the recombinant nucleic acid. In some embodiments, the nucleic analog is selected from the group consisting of 2’-O-methyl, 2’-O-methoxyethyl (2’-O-MOE), 2’-O-aminopropyl, 2’-deoxy, T- deoxy-2’ -fluoro, 2 ’-0 -aminopropyl (2’-O-AP), 2'-O-dimethylaminoethyl (2’-O-DMAOE), 2 -O- dimethylaminopropyl (2’-O-DMAP), T-O-dimethylaminoethyloxyethyl (2’-O-DMAEOE), 2’-O-N- methylacetamido (2’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2’-fluoro N3-P5’-phosphoramidite. In some embodiments, the recombinant nucleic acid molecule further comprises a leader sequence. In some embodiments, the recombinant nucleic acid molecule further comprises a promoter sequence. In some embodiments, the recombinant nucleic acid molecule further comprises a sequence encoding a poly(A) tail. In some embodiments, the recombinant nucleic acid molecule further comprises a 3’UTR sequence. In some embodiments, the recombinant nucleic acid molecule is an isolated nucleic acid or a non-naturally occurring nucleic acid. In some embodiments, the nucleic acid is an in vitro transcribed nucleic acid.

Recombinant Nucleic Acid Encoding HLA-E and/or HLA-G

[0353] Disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein and an additional nucleic acid sequence encoding an HLA-E or HLA-G polypeptide or a fragment thereof, or a fusion protein comprising HLA-E or HLA-G (e.g., B2M-HLA-E or B2M-HLA-G). Also disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein, a second nucleic acid sequence encoding a TCR constant domain described herein, and a third nucleic acid sequence encoding an HLA-E and/or HLA-G polypeptide or a fragment thereof, or a fusion protein comprising HLA-E and/or HLA-G. In some embodiments, the nucleic acid sequence encodes a B2M-HLA-E fusion protein. In some embodiments, the B2M-HLA-E fusion protein comprises a mutated B2M (e.g., SEQ ID NO: 420). In some embodiments, the B2M is mutated at the sgRNA binding site and PAM site to prevent cleavage by Cas9 during the generation of the B2M knockout cell in which the B2M-HLA-E fusion protein will be expressed. A mutated B2M is also referred to herein as “mB2M” or “mutB2M” and the like. In some embodiments, the B2M-HLA-E fusion protein comprises HLA- E*01:03 (e.g., SEQ ID NO: 422). In some embodiments, the B2M-HLA-E fusion protein comprises HLA- E*01:01. In some embodiments, the recombinant nucleic acid encodes both an HLA-E and an HLA-G; a fusion protein comprising an HLA-E and a fusion protein comprising an HLA-G; or a fusion protein comprising both an HLA-E and an HLA-G. Any recombinant nucleic acid molecules comprising a nucleic acid sequence encoding a TFP described herein may further comprise an additional nucleic acid sequence encoding an HLA-E and/or HLA-G polypeptide or a fragment thereof or fusion protein as described herein. The sequence encoding the TFP, the sequence encoding the TCR constant domain, and the sequence encoding an HLA-E and/or HLA-G polypeptide or fusion protein can be contained in the same or different nucleic acid molecules. For example, the sequence encoding the TFP, the sequence encoding the TCR constant domain, and the sequence encoding an HLA-E and/or HLA-G polypeptide or fusion protein can be contained in the same nucleic acid molecule. For another example, at least two of the sequences including the sequence encoding the TFP, the sequence encoding the TCR constant domain, and the sequence encoding an HLA-E and/or HLA-G polypeptide or fusion protein can be contained in the same nucleic acid molecule. For another example, one of the sequences including the sequence encoding the TFP, the sequence encoding the TCR constant domain, and the sequence encoding an HLA-E and/or HLA-G polypeptide or fusion protein can be contained in a separate nucleic acid molecule from the other two sequences.

[0354] Further disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein and an additional nucleic acid sequence encoding an HLA-E and/or HLA-G polypeptide or fusion protein, wherein the first nucleic acid sequence and the additional nucleic acid sequence are included in a single nucleic acid molecule. In some embodiments, the first nucleic acid sequence and the additional nucleic acid sequence are operatively linked by a first linker. For example, the first linker may be a cleavable linker. In some embodiments, the first 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 cleavage site can comprise a sequence of SEQ ID NO: 23, when expressed. In some embodiments, the first linker comprises a sequence of SEQ ID NO: 23, when expressed.

[0355] Further disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP described herein, a second nucleic acid sequence encoding a TCR constant domain described herein, and a third nucleic acid sequence encoding an HLA-E and/or HLA-G polypeptide or a fragment thereof, or a fusion protein comprising HLA-E and/or HLA-G; wherein the first, second, and third sequences are included in a single nucleic acid molecule. In some embodiments, the first, second, and third sequences are operatively linked by linkers, for example, a linker between the first and second sequence and a linker between the second and third sequence. In some embodiments, the first and second linkers 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 (e.g., SEQ ID NO: 23). In some embodiments, the protease cleavage site is a P2A cleavage site (e.g., SEQ ID NO: 365). In some embodiments, the first linker is a T2A and the second linker is a P2A. In some embodiments, the first linker is a P2A and the second linker is a T2A. [0356] In some embodiments, the nucleic acid sequence encoding the HLA-E and/or HLA-G polypeptide or fusion protein may comprise a sequence encoding a signal peptide. In some embodiments, the signal peptide may be a GMCSFR signal peptide or a B2M signal peptide. In some embodiments, the B2M signal peptide comprises a sequence of SEQ ID NO: 417.

[0357] In some embodiments, the nucleic acid encoding the B2M-HLA-E fusion protein comprises an HLA- G binding protein. In some embodiments, the HLA-G binding protein comprises a sequence according to SEQ ID NO: 418.

[0358] In some embodiments, the B2M-HLA-E fusion protein comprises a sequence having at least about 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 423. In some embodiments, the nucleic acid sequence encoding the B2M-HLA-E fusion protein may encode a sequence having at least about 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 423. In some embodiments, the fusion protein is expressed on a cell surface when expressed from the recombinant nucleic acid molecule described herein in a T cell. In some embodiments, the fusion protein comprises a sequence according to SEQ ID NO: 423.

[0359] The recombinant nucleic acid molecules that encode an HLA-E and/or HLA-G polypeptide or fusion protein can further comprise a sequence of any of the nucleic acid sequences listed in Table 4. In some embodiments, the recombinant nucleic acid molecules can encode an amino acid sequence of any of the amino acid sequences listed in Table 4. For example, the recombinant nucleic acid can comprise a sequence encoding a signal peptide. The signal peptide can be a GM-CSF signal peptide. The recombinant nucleic acid molecule can further comprise a sequence encoding a protease. The protease can be a furin. The recombinant nucleic acid can comprise a sequence of SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 400, SEQ ID NO: 402, and/or SEQ ID NO: 404. The recombinant nucleic acid molecule can comprise a sequence encoding SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 69, SEQ ID NO: 243, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, and/or SEQ ID NO: 21. The recombinant nucleic acid molecule can encode, from N-terminus to C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a linker (e.g., GSG linker), operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional anti-MSLN antigen binding domain, operatively linked to a TCR gamma constant domain. [0360] The recombinant nucleic acid molecule can encode, from N-terminus to C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a linker (e.g., GSG linker), operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional anti-MSLN antigen binding domain, operatively linked to a TCR gamma constant domain, operatively linked to a linker, operatively linked to a T2A sequence, operatively linked to a signal peptide (e.g., a B2M signal peptide), operatively linked to a HLA-G binding protein, operatively linked to a linker, operatively linked to a mutB2M, operatively linked to a linker, operatively linked to an HLA-E polypeptide. The recombinant nucleic acid molecule can encode, from N-terminus to C-terminus, a signal peptide (e.g., a B2M signal peptide), operatively linked to a HLA-G binding protein, operatively linked to a linker, operatively linked to a mutB2M, operatively linked to a linker, operatively linked to an HLA-E polypeptide, operatively linked to a T2A sequence, operatively linked to a GM-CSF signal peptide, operatively linked to an anti-MSLN antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a linker (e.g., GSG linker), operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional anti-MSLN antigen binding domain, operatively linked to a TCR gamma constant domain.

[0361] In some embodiments, the recombinant nucleic acid molecule is selected from the group consisting of a DNA and an RNA. In some embodiments, the recombinant nucleic acid molecule is an mRNA. In some embodiments, the recombinant nucleic acid molecule is a circRNA. In some embodiments, the recombinant nucleic acid molecule comprises a nucleic acid analog. In some embodiments, the nucleic acid analog is not in an encoding sequence of the recombinant nucleic acid. In some embodiments, the nucleic analog is selected from the group consisting of 2’-O-methyl, 2’-O-methoxyethyl (2’-0-M0E), 2’-O-aminopropyl, 2’-deoxy, T- deoxy-2’ -fluoro, 2 ’-0 -aminopropyl (2’-O-AP), 2'-O-dimethylaminoethyl (2’-0-DMA0E), 2 -O- dimethylaminopropyl (2’-0-DMAP), T-O-dimethylaminoethyloxyethyl (2’-0-DMAE0E), 2’-O-N- methylacetamido (2’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2’-fluoro N3-P5’-phosphoramidite. In some embodiments, the recombinant nucleic acid molecule further comprises a leader sequence. In some embodiments, the recombinant nucleic acid molecule further comprises a promoter sequence. In some embodiments, the recombinant nucleic acid molecule further comprises a sequence encoding a poly(A) tail. In some embodiments, the recombinant nucleic acid molecule further comprises a 3’UTR sequence. In some embodiments, the recombinant nucleic acid molecule is an isolated nucleic acid or a non-naturally occurring nucleic acid. In some embodiments, the nucleic acid is an in vitro transcribed nucleic acid.

Vectors

[0362] Further disclosed herein, in some embodiments, are vectors comprising the recombinant nucleic acid molecules disclosed herein. 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.

[0363] 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.

[0364] 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 long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0365] In another embodiment, the vector comprising the nucleic acid encoding the desired TFP, the constant domain, HLA-E and/or HLA-G fusion protein, IL- 15 polypeptide, and/or IL-15Ra polypeptide of the present disclosure is an adenoviral vector (A5/35). In another embodiment, the expression of nucleic acids encoding TFPs, the constant domain, HLA-E and/or HLA-G fusion protein, IL-15 polypeptide, and/or IL-15Ra polypeptide can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See below June et al. 2009 Nature Reviews Immunology 9.10: 704-716, is incorporated herein by reference.

[0366] 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, incorporated by reference herein in their entireties). In another embodiment, the present disclosure provides a gene therapy vector.

[0367] 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.

[0368] 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).

[0369] 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.

[0370] 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.

[0371] An example of a promoter that is capable of expressing a TFP transgene, constant domain transgene, HLA-E and/or HLA-G fusion protein transgene, IL-15 transgene, and/or IL-15Ra 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, IL-15, and/or IL-15Ra 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. [0372] In order to assess the expression of a TFP polypeptide, constant domain, HLA-E and/or HLA-G fusion protein, IL-15 polypeptide, and/or IL-15Ra 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.

[0373] 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.

[0374] 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.

[0375] 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

[0376] 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.

[0377] 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.

[0378] 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.

[0379] 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 selfrearrangement 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.

[0380] 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.

[0381] The present disclosure further provides a vector comprising a nucleic acid molecule encoding a TFP described herein, an HLA-E or HLA-G polypeptide or fusion protein described herein, an IL- 15 polypeptide or a fragment described herein, and/or IL-15Ra polypeptide or a fragment described herein. In one aspect, a vector encoding a TFP described herein, an HLA-E or HLA-G polypeptide or fusion protein described herein, an IL- 15 polypeptide or a fragment described herein, and/or IL-15Ra polypeptide or a fragment described herein 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, the HLA-E or HLA-G construct, the IL- 15 construct, and/or the IL-15Ra construct in mammalian T cells. In one aspect, the mammalian T cell is a human T cell. The present disclosure further provides compositions and methods wherein nucleic acid molecules encoding a TFP and constant domain, HLA-E and/or HLA-G fusion protein, and/or IL- 15 construct, and/or IL-15Ra constructs provided herein, are transduced into a cell. In some embodiments, these components are co-transduced into cells using two or more lentiviruses.

Recombinant RNAs

[0382] Disclosed herein are methods for producing in vitro transcribed RNA encoding TFPs, TCR constant domain, HLA-E, HLA-G, IL-15, and/or IL-15Ra described herein. The present disclosure also includes a TFP encoding RNA construct, a TCR constant domain encoding RNA construct, an HLA-E polypeptide or fusion protein encoding RNA construct, an HLA-G polypeptide or fusion protein encoding RNA construct, a IL- 15 encoding RNA construct, and/or IL-15Ra 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, HLA-E polypeptide or fusion protein, HLA-G polypeptide or fusion protein, IL- 15 polypeptide or a fragment thereof, and/or IL- 15Ra polypeptide or a fragment thereof described herein.

[0383] In one aspect the anti-TAA TFP, a TCR constant domain, HLA-E and/or HLA-G polypeptide or fusion protein, IL-15 polypeptide or a fragment thereof, and/or IL-15Ra polypeptide or a fragment thereof described herein is encoded by a messenger RNA (mRNA). In one aspect the mRNA encoding the anti-TAA TFP, HLA-E and/or HLA-G polypeptide or fusion protein, IL- 15 polypeptide or a fragment thereof, or IL- 15Ra polypeptide and/or a fragment thereof described herein is introduced into a T cell for production of a T cell expressing the TFP, HLA-E and/or HLA-G polypeptide or fusion protein, IL- 15 polypeptide or a fragment thereof, and/or IL-15Ra polypeptide or a fragment thereof described herein. In one embodiment, the in vitro transcribed RNA encoding a TFP, HLA-E and/or HLA-G polypeptide or fusion protein, IL- 15 polypeptide or a fragment thereof, or IL-15Ra polypeptide or a fragment thereof described herein 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.

[0384] 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. [0385] 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.

[0386] 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 3000 nucleotides in length. The length of 5 ’ and 3 ’ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5’ and 3’ UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

[0387] 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.

[0388] 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 do not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments the 5’ UTR can be 5 ’UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.

[0389] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription can 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. [0390] In some embodiments, 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 concatemeric 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. [0391] 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).

[0392] 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.

[0393] The polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a poly-T tail, such as 100 T tail (size can be 50-5000 T), 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.

[0394] 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.

[0395] 5’ caps can also provide stability to RNA molecules. In some embodiments, 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)).

[0396] 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.

[0397] 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® (Eppendorf, 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 Then, 12(8):861-70 (2001).

Modified Cells

[0398] Provided herein is a modified cell comprising a recombinant nucleic acid comprising a first sequence encoding a T cell receptor (TCR) fusion protein (TFP). The TFP can comprise a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, and (2) a TCR transmembrane domain, and an antibody domain comprising an antigen binding domain. The TCR subunit and the antibody can be operatively linked. The TFP can functionally incorporate into an endogenous TCR complex when expressed in the modified cell. The modified cell can comprise a functional disruption of an endogenous major histocompatibility complex (MHC) molecule. The modified cell can comprise an enhancing agent or a sequence encoding the enhancing agent that enhances persistence of the modified cell. The enhancing agent can comprise an interleukin- 15 (IL- 15) polypeptide or a fragment thereof. The modified cell can comprise a polypeptide or fusion protein to reduce NK cell lysis, for example, a B2M-HLA-E or B2M-HLA-G fusion protein, or both a B2M-HLA-E and a B2M-HLA-G fusion protein. The modified cell can comprise an IL- 15 polypeptide or fragment thereof and a polypeptide or fusion protein to reduce NK cell lysis, for example, a B2M-HLA-E or B2M-HLA-G fusion protein, or both a B2M-HLA-E and a B2M-HLA-G fusion protein. The endogenous MHC molecule can comprise all endogenous MHC molecules within the modified cell. The endogenous MHC molecule can comprise an MHC class I molecule, a MHC class II molecule, or a combination thereof. The functional disruption of the MHC molecule can comprise inactivating a gene encoding the MHC molecule or subunit thereof. The gene encoding the MHC molecule or subunit thereof can comprise knocking out or knocking down the gene. The gene encoding the MHC molecule or subunit thereof can comprise a gene encoding a beta-2 -microglobulin (B2M) molecule. In some cases, all endogenous MHC molecules in the modified cells are disrupted. The modified cell may not express any MHC molecules on a surface of the modified cell.

[0399] The functional disruption of the MHC molecule can comprise inactivating a gene encoding the MHC molecule or subunit thereof by various methods described herein. The inactivation can include disruption of genomic gene locus, gene silencing, inhibition or reduction of transcription, or inhibition or reduction of translation. The endogenous gene can be silenced, for example, by inhibitory nucleic acids such as siRNA and shRNA. The translation of the endogenous gene can be inhibited by inhibitory nucleic acids such as microRNA. In some embodiments, gene editing techniques are employed to disrupt an endogenous gene.

[0400] The TFP of the modified cell can further comprise a TCR intracellular domain. In some cases, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. For example, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR alpha, TCR beta, TCR gamma, TCR delta, CD3 epsilon, CD3 delta, or CD3 gamma. In some cases, all three of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon, CD3 delta, or CD3 gamma.

[0401] The recombinant nucleic acid of the modified cell can comprise a second sequence encoding a TCR constant domain. The TCR constant domain can be a TCR gamma constant domain or a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain. The second sequence can further encode a TCR transmembrane domain. The TCR transmembrane domain can be a TCR gamma transmembrane domain or a TCR delta transmembrane domain. The first sequence and the second sequence can be contained in a same recombinant nucleic acid molecule. In such cases, the recombinant nucleic acid molecule can further comprise a sequence encoding a protease cleavage site. 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. The first sequence and the second sequence can be contained in two separate recombinant nucleic acid molecules. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR alpha. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can comprise the constant domain of TCR alpha. The constant domain of TCR alpha may be murine constant domain. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can further comprise a TCR alpha transmembrane domain and a TCR alpha intracellular domain. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain may not comprise a variable domain of TCR alpha. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR beta. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can comprise the constant domain of TCR beta. The constant domain of TCR beta can be murine constant domain. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can further comprise a TCR beta transmembrane domain and a TCR beta intracellular domain. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain may not comprise a variable domain of TCR beta. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR gamma. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can comprise the constant domain of TCR gamma. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can further comprise a TCR gamma transmembrane domain and a TCR gamma intracellular domain. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain may not comprise a variable domain of TCR gamma. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR delta. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can comprise the constant domain of TCR delta. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can further comprise a TCR delta transmembrane domain and a TCR delta intracellular domain. The TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain may not comprise a variable domain of TCR delta. [0402] The modified cell described herein can comprise the NK cell inhibitor agent described herein. The modified cell can comprise a sequence encoding the NK cell inhibitor agent. The recombinant nucleic acid molecule described herein can comprise a third sequence that is the sequence encoding the NK cell inhibitor agent. The first sequence and the third sequence can be operatively linked by a first linker. The first linker can comprise a protease cleavage site. The protease cleavage site can be a 2A cleavage site. The NK cell inhibitor agent can comprise HLA-E or HLA-G. The NK cell inhibitor agent can comprise a mutated B2M fused to HLA-E, optionally further comprising an HLA-G binding protein.

[0403] The modified cell described herein can comprise the enhancing agent described herein. The modified cell can comprise a sequence encoding the enhancing agent. The recombinant nucleic acid molecule described herein can comprise a third sequence that is the sequence encoding the enhancing agent. The first sequence and the third sequence can be operatively linked by a first linker. The first linker can comprise a protease cleavage site. The protease cleavage site can be a 2A cleavage site. The enhancing agent can comprise an interleukin- 15 (IL-15) polypeptide or a fragment thereof. The IL-15 polypeptide may be secreted. The third sequence can further encode an IL- 15 receptor (IL-15R) subunit or a fragment thereof. The IL-15R subunit can be IL-15R alpha (IL-15Ra). The IL- 15 and IL-15Ra can be operatively linked by a second linker. The second linker may not be a cleavable linker. The second linker can comprise a sequence comprising (G4S)_n, wherein G is glycine, S is serine, and n is an integer from 1 to 10. Lor example, n can be an integer from 1 to 4. In some case, n is 3. The second linker can comprise a sequence of SEQ ID NO: 378 or 405.

[0404] The third sequence can encode a fusion protein comprising the IL- 15 polypeptide linked to the IL- 15Ra subunit. The IL-15 polypeptide can be linked to N-terminus of the IL-15Ra subunit. The fusion protein can comprise amino acids 30 - 162 of IL-15. The fusion protein can comprise amino acids 31 - 267 of IL- 15Ra. The fusion protein can further comprise a sushi domain. The fusion protein can comprise a sequence of SEQ ID NO: 389. In some cases, the fusion protein comprises a sequence of SEQ ID NO: 371. The fusion protein can be expressed on cell surface of the modified cell. The fusion protein can be secreted.

[0405] Also provided herein is a modified cell comprising a recombinant nucleic acid comprising a first sequence encoding a T cell receptor (TCR) fusion protein (TEP). The TFP can comprise a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, and (2) a TCR transmembrane domain, and an antibody domain comprising an antigen binding domain. The modified cell can further comprise a second sequence encoding a TCR constant domain described herein. For example, the TCR constant domain can be a TCR gamma constant domain or a TCR delta constant domain. In some cases, the modified cell can further comprise a second sequence encoding a TCR gamma constant domain and a TCR delta constant domain. The TCR subunit and the antibody can be operatively linked. The TFP can functionally incorporate into an endogenous TCR complex when expressed in a cell (e.g., the modified cell). The TCR extracellular domain and the TCR transmembrane domain can be from a same subunit. The same subunit can be TCR gamma or TCR delta. The TCR subunit can further comprise a TCR intracellular domain. The TCR intracellular domain can be from TCR gamma or TCR beta. The TCR extracellular domain, the TCR transmembrane domain and the TCR intracellular domain can be from a same subunit. The second sequence can further encode a second antibody domain comprising a second antigen binding domain. The second antigen binding domain and the antigen binding domain can be the same.

[0406] The modified cell can comprise a functional disruption of an endogenous major histocompatibility complex (MHC) molecule. The endogenous MHC molecule can comprise all endogenous MHC molecules within the modified cell. The endogenous MHC molecule can comprise an MHC class I molecule, a MHC class II molecule, or a combination thereof. The functional disruption of the MHC molecule can comprise inactivating a gene encoding the MHC molecule or subunit thereof. In some cases, inactivating the gene encoding the MHC molecule or subunit thereof comprises knocking out or knocking down the gene. The gene encoding the MHC molecule or subunit thereof can comprise a gene encoding a beta-2 -microglobulin (B2M) molecule. The modified cell may not express any MHC molecules on a surface of the modified cell. [0407] The modified cell can comprise an enhancing agent or a sequence encoding the enhancing agent that enhances persistence of the modified cell. For example, the modified cell can comprise the enhancing agent. The modified cell can comprise the sequence encoding the enhancing agent. The recombinant nucleic acid molecule can comprise a third sequence that is the sequence encoding the enhancing agent. The enhancing agent can comprise an interleukin- 15 (IL- 15) polypeptide or a fragment thereof. The first sequence and the third sequence can be operatively linked by a first linker. The first linker can comprise a protease cleavage site. For example, the protease cleavage site can be a 2A cleavage site. The IL- 15 polypeptide can be secreted. The third sequence can further encode an IL- 15 receptor (IL-15R) subunit or a fragment thereof. The IL-15R subunit can be IL-15R alpha (IL-15Ra). The IL- 15 and IL-15Ra can be operatively linked by a second linker. The second linker may not be a cleavable linker. The second linker can comprise a sequence comprising (G4S)_n, wherein G is glycine, S is serine, and n is an integer from 1 to 10. For example, n is an integer from 1 to 4. In some cases, n is 3. The third sequence can encode a fusion protein comprising the IL- 15 polypeptide linked to the IL-15Ra subunit. The IL-15 polypeptide can be linked to N-terminus of the IL- 15 Ra subunit.

[0408] The antibody domain can be an antibody fragment. For example, the antibody fragment can be a scFv, a single domain antibody domain, a VH domain or a VL domain. The antigen binding domain can be selected from a group consisting of an anti-CD19 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, an anti-79b binding domain, an anti-HER2 binding domain, an anti-PMSA binding domain, an anti-MUC16 binding domain, an anti-CD22 binding domain, an anti-PD-Ll binding domain, an anti BAFF or BAFF receptor binding domain, an anti-Nectin-4 binding domain, an anti-TROP-2 binding domain, an anti- GPC3 binding domain, and an anti-ROR-1 binding domain.

[0409] Disclosed herein, in some embodiments, are cells comprising the recombinant nucleic acid disclosed herein, the polypeptide disclosed herein, or the vectors disclosed herein. Disclosed herein, in some embodiments, are cells comprising the recombinant nucleic acid disclosed herein, the polypeptide disclosed herein, or the vectors disclosed herein; wherein cells comprising the sequence encoding a TFP disclosed herein, an IL-15 polypeptide or a fragment disclosed herein, and/or an IL-15Ra polypeptide or a fragment disclosed herein.

[0410] In some embodiments, the cell is a T cell. In some embodiments, the T cell is a human T cell. In some embodiments, the T cell is a CD8+ or CD4+ T cell. In some embodiments, the T cell is a human ot|3 T cell. In some embodiments, the T cell is a human y5 T cell. 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 y5 T cells and do not comprise a functional disruption of an endogenous TCR. In some embodiments, the y5 T cells are V51+ V52- y8 T cells. In some embodiments, the y5 T cells are V51- V52+ y8 T cells. In some embodiments, the y5 T cells are V51- V52- y8 T cells.

[0411] Disclosed herein, in some embodiments, are cells comprising the recombinant nucleic acid disclosed herein, the polypeptide disclosed herein, or the vectors disclosed herein wherein cells comprising the sequence encoding TFP disclosed herein, IL- 15 polypeptide or a fragment disclosed herein, and/or IL-15Ra polypeptide or a fragment disclosed herein. In some embodiments, the IL-15 polypeptide or a fragment thereof is secreted when expressed in a cell. For example, cells disclosed herein may secrete IL-15 polypeptide expressed from the recombinant nucleic acid molecules disclosed herein in response to a cell activation agent. In some embodiments, IL- 15 signaling is increased in response to a cell activation agent. In some embodiment, the cell activation agent comprises a T cell activation agent. A T cell activation agent, as described herein, may include, but is not limited to, an anti-CD3 antibody or a fragment thereof, an anti-CD28 antibody or a fragment thereof, a cytokine, an antigen that binds the antigen binding domain of the TFP described herein, or any combinations thereof.

[0412] Disclosed herein, in some embodiments, are cells comprising the sequence encoding TFP disclosed herein, IL- 15 polypeptide or a fragment disclosed herein, and/or IL-15Ra polypeptide or a fragment disclosed herein may have enhanced survival rate, enhanced effector function, and/or enhanced cytotoxicity compared to cells that do not comprise the sequence encoding TFP disclosed herein, IL- 15 polypeptide or a fragment disclosed herein, and/or IL-15Ra polypeptide or a fragment disclosed herein. In some embodiments, the cell has enhanced survival rate compared to a cell that does not have IL- 15 signaling. In some embodiments, the cell has enhanced survival rate compared to a cell that does not express the IL- 15 polypeptide or a fragment thereof and/or IL-15Ra polypeptide or a fragment thereof. In some embodiments, the cell has enhanced effector function compared to a cell that does not have IL-15 signaling. In some embodiments, the cell has enhanced effector function compared to a cell that does not express the IL- 15 polypeptide or a fragment thereof and/or IL-15Ra polypeptide or a fragment thereof. In some embodiments, the cell has enhanced cytotoxicity compared to a cell that does not have IL-15 signaling. In some embodiments, the cell has enhanced cytotoxicity compared to a cell that does not express the IL- 15 polypeptide or a fragment thereof and/or IL-15Ra polypeptide or a fragment thereof.

[0413] Disclosed herein, in some embodiments, are cells comprising the sequence encoding TFP disclosed herein, IL- 15 polypeptide or a fragment disclosed herein, and/or IL-15Ra polypeptide or a fragment disclosed herein may have increased longevity compared to cells that do not comprise the sequence encoding TFP disclosed herein, IL- 15 polypeptide or a fragment disclosed herein, and/or IL-15Ra polypeptide or a fragment disclosed herein. In some embodiments, the longevity of the cell is increased compared to a cell that does not comprise (i) a nucleic acid sequence encoding an interleukin- 15 (IL- 15) polypeptide or a fragment thereof or (ii) a nucleic acid sequence encoding an interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof.

[0414] Disclosed herein, in some embodiments, are cells comprising the sequence encoding TFP disclosed herein, IL- 15 polypeptide or a fragment disclosed herein, and/or IL-15Ra polypeptide or a fragment disclosed herein may have increased persistence compared to cells that do not comprise the sequence encoding TFP disclosed herein, IL- 15 polypeptide or a fragment disclosed herein, and/or IL-15Ra polypeptide or a fragment disclosed herein. In some embodiments, the persistence of the cell is increased compared to a cell that does not comprise (i) a nucleic acid sequence encoding an interleukin- 15 (IL- 15) polypeptide or a fragment thereof or (ii) a nucleic acid sequence encoding an interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof.

[0415] Disclosed herein, in some embodiments, are cells comprising the sequence encoding TFP disclosed herein, IL- 15 polypeptide or a fragment disclosed herein, and/or IL-15Ra polypeptide or a fragment disclosed herein may have increased cytotoxicity compared to cells that do not comprise the sequence encoding TFP disclosed herein, IL- 15 polypeptide or a fragment disclosed herein, and/or IL-15Ra polypeptide or a fragment disclosed herein. In some embodiments, the cytotoxicity of the cell is increased compared to a cell that does not comprise (i) a nucleic acid sequence encoding an interleukin- 15 (IL- 15) polypeptide or a fragment thereof or (ii) a nucleic acid sequence encoding an interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof.

[0416] Disclosed herein, in some embodiments, are cells comprising the sequence encoding TFP disclosed herein, IL- 15 polypeptide or a fragment disclosed herein, and/or IL-15Ra polypeptide or a fragment disclosed herein may have increased cytokine production compared to cells that do not comprise the sequence encoding TFP disclosed herein, IL- 15 polypeptide or a fragment disclosed herein, and/or IL-15Ra polypeptide or a fragment disclosed herein. In some embodiments, the cytokine production of the cell is increased compared to a cell that does not comprise (i) a nucleic acid sequence encoding an interleukin- 15 (IL- 15) polypeptide or a fragment thereof or (ii) a nucleic acid sequence encoding an interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof.

[0417] In some embodiments, cells disclosed herein retains naive and/or central memory phenotypes. In some embodiments, cells disclosed herein have not differentiated into terminal effector cells.

[0418] Disclosed herein, in some embodiments, is a population of cells comprising any of the cell described herein. Disclosed herein, in some embodiments, is a population of cells comprising any of the cell described herein, wherein the population of cells has an increased proportion of cells having a central memory phenotype relative to a population of cells that do not comprise the sequence encoding TFP disclosed herein, IL-15 polypeptide or a fragment disclosed herein, and/or IL-15Ra polypeptide or a fragment disclosed herein. In some embodiments, the population of cells has an increased proportion of cells having a central memory phenotype relative to a population of cells that do not comprise (i) a nucleic acid sequence encoding an interleukin- 15 (IL- 15) polypeptide or a fragment thereof or (ii) a nucleic acid sequence encoding interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof.

[0419] Disclosed herein, in some embodiments, is population of cells comprising any of the cell described herein, wherein the population of cells has an increased proportion of cells having a naive phenotype relative to a population of cells that do not comprise the sequence encoding TFP disclosed herein, IL- 15 polypeptide or a fragment disclosed herein, and/or IL-15Ra polypeptide or a fragment disclosed herein. In some embodiments, the population of cells has an increased proportion of cells having a naive phenotype relative to a population of cells that do not comprise (i) a nucleic acid sequence encoding an interleukin- 15 (IL- 15) polypeptide or a fragment thereof or (ii) a nucleic acid sequence encoding an interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof.

[0420] Disclosed herein, in some embodiments, is population of cells comprising any of the cell described herein, wherein the population of cells has a reduced proportion of cells having a terminal effector phenotype relative to a population of cells that do not comprise the sequence encoding TFP disclosed herein, IL- 15 polypeptide or a fragment disclosed herein, and/or IL-15Ra polypeptide or a fragment disclosed herein. In some embodiments, the population of cells has a reduced proportion of cells having a terminal effector phenotype relative to a population of cells that do not comprise (i) a nucleic acid sequence encoding an interleukin- 15 (IL- 15) polypeptide or a fragment thereof or (ii) a nucleic acid sequence encoding an interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof.

[0421] Disclosed herein, in some embodiments, are modified T cells comprising the recombinant nucleic acid disclosed herein, or the vectors disclosed herein; wherein the modified T cell comprises a functional disruption of an endogenous TCR. Also disclosed herein, in some embodiments, are modified T cells comprising the sequence encoding the TFP of the nucleic acid disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein, wherein the modified T cell comprises a functional disruption of an endogenous TCR. Further disclosed herein, in some embodiments, are modified allogenic T cells comprising the sequence encoding the TFP disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein.

[0422] 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, 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. In some instances, the endogenous TCR that is functionally disrupted is an endogenous TCR alpha chain, an endogenous TCR beta constant domain, an endogenous TCR alpha constant domain and an endogenous TCR beta constant domain, 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 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 embodiments, a T cell expressing the TFP descried herein can inhibit tumor growth when expressed in a T cell.

[0423] In some embodiments, proliferation of the cell is increased compared to a cell that does not comprise (i) a nucleic acid sequence encoding an interleukin- 15 (IL-15) polypeptide or a fragment thereof or (ii) a nucleic acid sequence encoding an interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof or (iii) a nucleic acid sequence encoding an NK cell inhibitor agent (e.g., HLA-E or HLA-G, for example, an NK cell inhibitor agent comprising a mutated B2M fused to HLA-E, optionally further comprising an HLA-G binding protein). For example, the proliferation of the cell can be increased by at least about 5%. In some embodiments, IL- 15 is operatively linked to IL-15Ra.

[0424] In some embodiments, the activity or persistence of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein is increased. In some embodiments, IL- 15 is operatively linked to IL-15Ra. In some embodiments, the activity or persistence of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%, at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least 10000%, at least 20000%, at least 30000%, at least 40000%, at least 50000%, at least 60000%, at least 70000%, at least 80000%, at least 90000%, or at least 100000% as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but that do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein. In some embodiments, the activity or persistence of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein is increased by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 450 fold, at least 500 fold, at least 550 fold, at least 600 fold, at least 650 fold, at least 700 fold, at least 750 fold, at least 800 fold, at least 850 fold, at least 900 fold, at least 950 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but that do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein.

[0425] In some embodiments, the activity or persistence of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein is increased. In some embodiments, the activity or persistence of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%, at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least 10000%, at least 20000%, at least 30000%, at least 40000%, at least 50000%, at least 60000%, at least 70000%, at least 80000%, at least 90000%, or at least 100000% as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein. In some embodiments, the activity or persistence of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein is increased by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold, at least

100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 450 fold, at least 500 fold, at least 550 fold, at least 600 fold, at least 650 fold, at least 700 fold, at least 750 fold, at least 800 fold, at least 850 fold, at least 900 fold, at least 950 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein.

[0426] In some embodiments, the activity or persistence of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a nucleic acid sequence encoding an NK cell inhibitor agent (e.g., HLA-E or HLA-G, for example, an NK cell inhibitor agent comprising a mutated B2M fused to HLA-E, optionally further comprising an HLA-G binding protein), is increased. In some embodiments, the activity or persistence of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding a nucleic acid sequence encoding an NK cell inhibitor as described herein is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%, at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least 10000%, at least 20000%, at least 30000%, at least 40000%, at least 50000%, at least 60000%, at least 70000%, at least 80000%, at least 90000%, or at least 100000% as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but that do not express a recombinant nucleic acid molecule comprising a sequence encoding a nucleic acid sequence encoding an NK cell inhibitor as described herein. In some embodiments, the activity or persistence of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding a nucleic acid sequence encoding an NK cell inhibitor agent (e.g., HLA-E or HLA-G, for example, an NK cell inhibitor agent comprising a mutated B2M fused to HLA-E, optionally further comprising an HLA-G binding protein) as described herein is increased by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 450 fold, at least 500 fold, at least 550 fold, at least 600 fold, at least 650 fold, at least 700 fold, at least 750 fold, at least 800 fold, at least 850 fold, at least 900 fold, at least 950 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but that do not express a recombinant nucleic acid molecule comprising a sequence encoding a nucleic acid sequence encoding an NK cell inhibitor agent as described herein. [0427] In some embodiments, the proliferation of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein is increased. In some embodiments, IL- 15 is operatively linked to IL- 15Ra. In some embodiments, the proliferation of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%, at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least 10000%, at least 20000%, at least 30000%, at least 40000%, at least 50000%, at least 60000%, at least 70000%, at least 80000%, at least 90000%, or at least 100000% as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein. In some embodiments, the proliferation of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein is increased by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 450 fold, at least 500 fold, at least 550 fold, at least 600 fold, at least 650 fold, at least 700 fold, at least 750 fold, at least 800 fold, at least 850 fold, at least 900 fold, at least 950 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein.

[0428] In some embodiments, the proliferation of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein is increased. In some embodiments, the proliferation of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%, at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least 10000%, at least 20000%, at least 30000%, at least 40000%, at least 50000%, at least 60000%, at least 70000%, at least 80000%, at least 90000%, or at least 100000% as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein. In some embodiments, the proliferation of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein is increased by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 450 fold, at least 500 fold, at least 550 fold, at least 600 fold, at least 650 fold, at least 700 fold, at least 750 fold, at least 800 fold, at least 850 fold, at least 900 fold, at least 950 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein.

[0429] In some embodiments, the proliferation of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an NK cell inhibitor agent (e.g., HLA-E or HLA-G, for example, an NK cell inhibitor agent comprising a mutated B2M fused to HLA- E, optionally further comprising an HLA-G binding protein), as described herein is increased. In some embodiments, the proliferation of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an NK cell inhibitor agent as described herein is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%, at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least 10000%, at least 20000%, at least 30000%, at least 40000%, at least 50000%, at least 60000%, at least 70000%, at least 80000%, at least 90000%, or at least 100000% as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but that do not express a recombinant nucleic acid molecule comprising a sequence encoding a NK cell inhibitor agent as described herein. In some embodiments, the proliferation of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an NK cell inhibitor agent (e.g., HLA-E or HLA-G, for example, an NK cell inhibitor agent comprising a mutated B2M fused to HLA-E, optionally further comprising an HLA-G binding protein) as described herein is increased by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 450 fold, at least 500 fold, at least 550 fold, at least 600 fold, at least 650 fold, at least 700 fold, at least 750 fold, at least 800 fold, at least 850 fold, at least 900 fold, at least 950 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but that do not express a recombinant nucleic acid molecule comprising a sequence encoding an NK cell inhibitor agent as described herein.

[0430] In some embodiments, expression of an exhaustion marker of the cell is decreased compared to a cell that does not comprise (i) a nucleic acid sequence encoding an interleukin- 15 (IL-15) polypeptide or a fragment thereof or (ii) a nucleic acid sequence encoding an interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof. For example, expression of the exhaustion marker of the cell can be decreased for at least about 5% . The exhaustion marker can be PD-1, TIM-3 or LAG-3 . In some embodiments, IL- 15 is operatively linked to IL-15Ra.

[0431] In some embodiments, the expression of one or more exhaustion markers in the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15 polypeptide or a fragment thereof as described herein is decreased. In some embodiments, IL- 15 is operatively linked to IL-15Ra. In some embodiments, the expression of one or more exhaustion markers in the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein is decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein. In some embodiments, the expression of one or more exhaustion markers in the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein is decreased by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 450 fold, at least 500 fold, at least 550 fold, at least 600 fold, at least 650 fold, at least 700 fold, at least 750 fold, at least 800 fold, at least 850 fold, at least 900 fold, at least 950 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein.

[0432] In some embodiments, the expression of one or more exhaustion markers in the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein is decreased. In some embodiments, the expression of one or more exhaustion markers in the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein is decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein. In some embodiments, the expression of one or more exhaustion markers in the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein is decreased by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 450 fold, at least 500 fold, at least 550 fold, at least 600 fold, at least 650 fold, at least 700 fold, at least 750 fold, at least 800 fold, at least 850 fold, at least 900 fold, at least 950 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein.

[0433] In some embodiments, expression of TCF-1 of the cell is increased compared to a cell that does not comprise (i) a nucleic acid sequence encoding an interleukin- 15 (IL- 15) polypeptide or a fragment thereof or (ii) a nucleic acid sequence encoding an interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof. For example, the expression of TCF-1 of the cell is increased for at least about 5%. In some embodiments, IL- 15 is operatively linked to IL-15Ra.

[0434] In some embodiments, the TCF-1 + T cell population is increased in a population of the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein. In some embodiments, IL-15 is operatively linked to IL-15Ra. In some embodiments, the TCF-1+ T cell population is increased in a population of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%, at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least 10000%, at least 20000%, at least 30000%, at least 40000%, at least 50000%, at least 60000%, at least 70000%, at least 80000%, at least 90000%, or at least 100000% as compared with a population of the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL-15 polypeptide or a fragment thereof as described herein. In some embodiments, the TCF-1+ T cell population is increased in a population of the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 450 fold, at least 500 fold, at least 550 fold, at least 600 fold, at least 650 fold, at least 700 fold, at least 750 fold, at least 800 fold, at least 850 fold, at least 900 fold, at least 950 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared with a population of the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein.

[0435] In some embodiments, the TCF-1+ T cell population is increased in a population of the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein. In some embodiments, the TCF-1+ T cell population is increased in a population of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%, at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least 10000%, at least 20000%, at least 30000%, at least 40000%, at least 50000%, at least 60000%, at least 70000%, at least 80000%, at least 90000%, or at least 100000% as compared with a population of the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein. In some embodiments, the TCF-1+ T cell population is increased in a population of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 450 fold, at least 500 fold, at least 550 fold, at least 600 fold, at least 650 fold, at least 700 fold, at least 750 fold, at least 800 fold, at least 850 fold, at least 900 fold, at least 950 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared with a population of the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein.

[0436] In some embodiments, tumor infiltration of the cell is increased compared to a cell that does not comprise (i) a nucleic acid sequence encoding an interleukin- 15 (IL- 15) polypeptide or a fragment thereof or (ii) a nucleic acid sequence encoding an interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof. For example, the tumor infiltration of the cell can be increased for at least about 2-fold. In some embodiments, IL- 15 is operatively linked to IL-15Ra. In some embodiments, the tumor infdtration of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein is increased. In some embodiments, IL- 15 is operatively linked to IL-15Ra. In some embodiments, the tumor infdtration of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%, at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least 10000%, at least 20000%, at least 30000%, at least 40000%, at least 50000%, at least 60000%, at least 70000%, at least 80000%, at least 90000%, or at least 100000% as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein. In some embodiments, the tumor infdtration of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein is increased by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 450 fold, at least 500 fold, at least 550 fold, at least 600 fold, at least 650 fold, at least 700 fold, at least 750 fold, at least 800 fold, at least 850 fold, at least 900 fold, at least 950 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein.

[0437] In some embodiments, the tumor infdtration of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein is increased. In some embodiments, the tumor infdtration of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%, at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least 10000%, at least 20000%, at least 30000%, at least 40000%, at least 50000%, at least 60000%, at least 70000%, at least 80000%, at least 90000%, or at least 100000% as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein. In some embodiments, the tumor infiltration of the cell expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein and a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein is increased by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 450 fold, at least 500 fold, at least 550 fold, at least 600 fold, at least 650 fold, at least 700 fold, at least 750 fold, at least 800 fold, at least 850 fold, at least 900 fold, at least 950 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared with the cells expressing a recombinant nucleic acid molecule comprising a sequence encoding TFP as described herein, but do not express a recombinant nucleic acid molecule comprising a sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein.

Sources of T cells

[0438] Prior to expansion and genetic modification, a source of T cells is obtained from a subject. The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects of the present 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 Oncology CytoMate™, or the Haemonetics® Cell Saver® 5) according to the manufacturer’s instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture media.

[0439] In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL® gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one aspect, T cells are isolated by incubation with anti-CD3/anti-CD28 (e.g., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In one aspect, the time period is about 30 minutes. In a further aspect, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti- CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this 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.

[0440] 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.

[0441] 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.: WO 2013/126712.

[0442] 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.

[0443] 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 5xlO⁶/mL. In other aspects, the concentration used can be from about I x I OVmL to lxlO⁶/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.

[0444] 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.

[0445] 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 can 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, and mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, and irradiation.

[0446] 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

[0447] 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. [0448] Generally, the T cells of the present disclosure may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells, 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). 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 gamma-chain (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.

[0449] 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-1 -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. [0450] T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.

[0451] Further, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.

[0452] Once an anti-CD19, anti-BCMA, anti-CD22, anti-RORl, anti-PD-1, or anti-BAFF, anti-MUC16, anti- mesothelin, anti-HER2, anti-PMSA, anti-CD20, anti-CD70, anti-GPC3, anti-Nectin-4, anti-Trop2, or antiCD79b 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-CD19, anti-BCMA, anti-GPC3, anti-Nectin-4, anti-Trop2, anti-CD22, anti- MSLN, anti-CD79B, anti-RORl, anti-PD-1, anti-IL13Ra2, anti-PD-Ll, anti-CD20, anti-CD70, or anti-BAFF or BAFFR TFP are described in further detail below.

[0453] 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.

[0454] In vitro expansion of TFP⁺T cells following antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4⁺ and CD8⁺ T cells are stimulated with alphaCD3/alphaCD28 and APCs followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed. Exemplary promoters include the CMV IE gene, EF-lalpha, ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescence is evaluated on day 6 of culture in the CD4+ and/or CD8+ T cell subsets by flow cytometry (see, e.g, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Alternatively, a mixture of CD4+ and CD8+ T cells are stimulated with alphaCD3/alphaCD28 coated magnetic beads on day 0 and transduced with TFP on day 1 using a bicistronic lentiviral vector expressing TFP along with eGFP using a 2A ribosomal skipping sequence. 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)).

[0455] Sustained TFP+ T cell expansion in the absence of re -stimulation can also be measured (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer III particle counter following stimulation with alphaCD3/alphaCD28 coated magnetic beads on day 0, and transduction with the indicated TFP on day 1. [0456] Animal models can also be used to measure a TFP-T activity. For example, xenograft model using, e.g., human CD19-specific TFP+ T cells to treat a primary human pre-B ALL in immunodeficient mice can be used (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). After establishment of ALL, 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 B-ALL. 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 leukemia at weekly intervals. Peripheral blood CD 19+ B-ALL blast cell counts are measured in mice that are injected with alphaCD 19-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 leukemic cells and 3 weeks later are injected with T cells engineered to express TFP by a bicistronic lentiviral vector that encodes the TFP linked to eGFP. T cells are 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 leukemia at 1-week intervals. Survival curves for the TFP+ T cell groups are compared using the log-rank test.

[0457] 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 leukemia 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 CD 19+ ALL blast counts and then killed on days 35 and 49. The remaining animals are evaluated on days 57 and 70.

[0458] 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 K562 cells expressing the tumor associated antigen (TAA, e.g., CD19) CD19 (K19) or CD32 and CD137 (KT32-BBL) for a final T cell:K562 ratio of 2: 1. K562 cells are irradiated with gamma-radiation prior to use. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultures with KT32-BBL cells to serve as a positive control for stimulating T cell proliferation since these signals support long-term CD8+ T cell expansion ex vivo. T cells are enumerated in cultures using CountBright™ fluorescent beads (Invitrogen) and flow cytometry as described by the manufacturer. TFP+ T cells are identified by GFP expression using T cells that are engineered with eGFP-2A linked TFP -expressing lentiviral vectors. For TFP+ T cells not expressing GFP, the TFP+ T cells are detected with biotinylated recombinant CD19 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 restimulation 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.

[0459] Cytotoxicity can be assessed by a standard ⁵¹Cr-release assay (see, e.g. , Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Target cells (K562 lines and primary pro-B-ALL cells) are loaded with ⁵¹Cr (as NaCrCE, 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 cell: target 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 ⁵¹Cr 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 ⁵¹Cr released for each experimental condition.

[0460] Imaging technologies can be used to evaluate specific trafficking and proliferation of TFPs in tumorbearing animal models. Such assays have been described, e.g., in Barrett et al., Human Gene Therapy T. 1575-1586 (2011). NOD/SCID/yc-/- (NSG) mice are injected IV with Nalm-6 cells (ATCC® CRL- 3273™) followed 7 days later with T cells 4 hour after electroporation with the TFP constructs. The T cells are stably transfected with a lentiviral construct to express firefly luciferase, and mice are imaged for bioluminescence. Alternatively, therapeutic efficacy and specificity of a single injection of TFP+ T cells in Nalm-6 xenograft model can be measured as the following: NSG mice are injected with Nalm-6 transduced to stably express firefly luciferase, followed by a single tail-vein injection of T cells electroporated with a TAA- TFP 7 days later. Animals are imaged at various time points post injection. For example, photon-density heat maps of firefly luciferase positive leukemia in representative mice at day 5 (2 days before treatment) and day 8 (24 hours post TFP+ PBLs) can be generated.

[0461] 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-CD19, anti-BCMA, anti-CD22, anti-MSLN, anti-CD79B, anti- GPC3, anti-Nectin-4, anti-Trop2, anti-IL13Ra2, anti-PD-1, anti-RORl, anti-PD-Ll, or anti-BAFF or BAFFR TFP constructs disclosed herein. Pharmaceutical Compositions

[0462] Disclosed herein, in some embodiments, are pharmaceutical compositions comprising: (a) the cells of the disclosure; and (b) a pharmaceutically acceptable carrier. Disclosed herein, in some embodiments, are pharmaceutical compositions comprising: (a) the modified cells (e.g., modified T cells) of the disclosure; and (b) a pharmaceutically acceptable carrier. 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.

[0463] 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.

[0464] 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.

[0465] When “an immunologically effective amount,” “an anti-tumor effective amount,” “a tumor-inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, in some instances 10⁵ to 10⁶ 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. Med. 319: 1676, 1988).

[0466] 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.

-I l l- [0467] 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.

[0468] 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 modified T-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 modified T cells of the present disclosure. In an additional aspect, expanded cells are administered before or following surgery.

[0469] 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, 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).

[0470] In one embodiment, the TFP is introduced into T cells, e.g., using in vitro transcription, and the subject (e.g, human) receives an initial administration of TFP T cells of the present disclosure, and one or more subsequent administrations of the TFP T cells of the present disclosure, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, more than one administration of the TFP T cells of the present disclosure are administered to the subject (e.g., human) per week, e.g., 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. [0471] In one aspect, MSLN TFP T cells are generated using lentiviral viral vectors, such as lentivirus. TFP- T cells generated that way will have stable TFP expression.

[0472] 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 affected by RNA TFP vector delivery. In one aspect, the TFP RNA is transduced into the T cell by electroporation.

[0473] 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.

[0474] 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.

[0475] 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.

Methods of Producing Modified Cells

[0476] Provided herein, in some cases, is a method of producing the modified cell described herein. The method can comprise functionally disrupting an endogenous MHC molecule of a cell. Next, the cell containing a functional disruption of the endogenous MHC gene can be transduced with the recombinant nucleic acid described herein. The recombinant nucleic acid molecule can comprise a sequence encoding the TFP, a sequence encoding a constant domain, and/or a sequence encoding an enhancing agent. In some cases, the method can further comprise functionally disrupting an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain; thereby producing a cell containing a functional disruption of an endogenous TCR gene. Disrupting the endogenous TCR gene can comprise transducing the cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets the endogenous gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.

[0477] Also provided herein is a method of producing the modified cell described herein. The method can comprise transducing a cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid described herein. The cell containing a functional disruption of an endogenous TCR gene can be a cell containing a functional disruption of an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain. The cell can further comprise a functional disruption of an endogenous MHC molecule. The cell can comprise a functional disruption of a gene encoding a B2M molecule. The cell can be a T cell. The T cell can be a human T cell. The cell containing a functional disruption of an endogenous TCR gene can have reduced binding to MHC-peptide complex compared to that of an unmodified control cell. Functional disruption of endogenous genes encoding TCR chains or MHC molecules can be performed by various methods including the gene editing methods described herein. For example, disrupting the endogenous gene can comprise transducing the cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets the endogenous gene. The nuclease protein can be a meganuclease, a zinc -finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, or a megaTAL nuclease. The sequence comprised by the recombinant nucleic acid can be inserted into the endogenous TCR subunit gene at the cleavage site. The insertion of the sequence into the endogenous TCR subunit gene can functionally disrupt the endogenous TCR subunit. The nuclease protein can be a meganuclease. The meganuclease can comprise a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence, and wherein the second subunit binds to a second recognition half-site of the recognition sequence. The meganuclease can be a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.

[0478] Disclosed herein, in some embodiments, are methods of producing the modified T cell of the disclosure, the method comprising (a) disrupting an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, or any combination thereof; thereby producing a T cell containing a functional disruption of an endogenous TCR gene; and (b) transducing the T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid of the disclosure, or the vectors disclosed herein. In some instances, disrupting comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets the endogenous gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.

[0479] Further disclosed herein, in some embodiments, are methods of producing the modified T cell of the disclosure, the method comprising transducing a T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid disclosed herein, or the vectors disclosed herein. In some instances, the T cell containing a functional disruption of an endogenous TCR gene is a T cell containing a functional disruption of an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.

[0480] In some instances, the T cell is a human T cell. In some instances, the T cell containing a functional disruption of an endogenous TCR gene has reduced binding to MHC-peptide complex compared to that of an unmodified control T cell.

[0481] In some instances, the nuclease is a meganuclease, a zinc -finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, CRISPR/Cas nickase, or a megaTAL nuclease. In some instances, the sequence comprised by the recombinant nucleic acid or the vector is inserted into the endogenous TCR subunit gene at the cleavage site, and wherein the insertion of the sequence into the endogenous TCR subunit gene functionally disrupts the endogenous TCR subunit. In some instances, the nuclease is a meganuclease. In some instances, the meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence, and wherein the second subunit binds to a second recognition half-site of the recognition sequence. In some instances, the meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.

Gene Editins Technologies

[0482] In some embodiments, the modified T cells disclosed herein are engineered using a gene editing technique such as clustered regularly interspaced short palindromic repeats (CRISPR®, see, e.g., US Patent No. 8,697,359), transcription activator-like effector (TALE) nucleases (TALENs, see, e.g., U.S. Patent No. 9,393,257), meganucleases (endodeoxyribonucleases having large recognition sites comprising doublestranded DNA sequences of 12 to 40 base pairs), zinc finger nuclease (ZFN, see, e.g., Umov et al., Nat. Rev. Genetics (2010) vl 1, 636-646), or megaTAL nucleases (a fusion protein of a meganuclease to TAL repeats) methods. In this way, a chimeric construct may be engineered to combine desirable characteristics of each subunit, such as conformation or signaling capabilities. See also Sander & Joung, Nat. Biotech. (2014) v32, 347-55; and June et al., 2009 Nature Reviews Immunol. 9.10: 704-716, each incorporated herein by reference. In some embodiments, one or more of the extracellular domain, the transmembrane domain, or the cytoplasmic domain of a TFP subunit are engineered to have aspects of more than one natural TCR subunit domain (i.e., are chimeric).

[0483] Recent developments of technologies to permanently alter the human genome and to introduce sitespecific genome modifications in disease relevant genes lay the foundation for therapeutic applications. These technologies are now commonly known as “genome editing.”

[0484] The endogenous gene encoding a major histocompatibility complex (MHC) molecule can be disrupted in the modified cells described herein. The endogenous MHC molecule can comprise all endogenous MHC molecules within the modified cell. The endogenous MHC molecule can comprise an MHC class I molecule, a MHC class II molecule, or a combination thereof. The functional disruption of the MHC molecule can comprise inactivating a gene encoding the MHC molecule or subunit thereof. The inactivation can include disruption of genomic gene locus, gene silencing, inhibition or reduction of transcription, or inhibition or reduction of translation. The endogenous gene can be silenced, for example, by inhibitory nucleic acids such as siRNA and shRNA. The translation of the endogenous gene can be inhibited by inhibitory nucleic acids such as microRNA. In some embodiments, gene editing techniques are employed to disrupt an endogenous gene. In some cases, inactivating the gene encoding the MHC molecule or subunit thereof can comprise knocking out or knocking down the gene. The gene encoding the MHC molecule or subunit thereof can comprise a gene encoding a beta-2 -microglobulin (B2M) molecule.

[0485] The endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain can be inactivated in the modified cell (e.g., modified T cell) described herein. The inactivation can include disruption of genomic gene locus, gene silencing, inhibition or reduction of transcription, or inhibition or reduction of translation. The endogenous TCR gene can be silenced, for example, by inhibitory nucleic acids such as siRNA and shRNA. The translation of the endogenous TCR gene can be inhibited by inhibitory nucleic acids such as microRNA. In some embodiments, gene editing techniques are employed to disrupt an endogenous TCR gene. In some embodiments, mentioned endogenous TCR gene encodes a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain. In some embodiments, gene editing techniques pave the way for multiplex genomic editing, which allows simultaneous disruption of multiple genomic loci in endogenous TCR gene. In some embodiments, multiplex genomic editing 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 programmed cell death protein 1 (PD- 1), and/or other genes.

[0486] 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.

[0487] 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 base pairs 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 atandem array of TAL- effector domains, each of which specifically recognizes a single DNA base pair. Compact TALENs have an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley et al. (2013), Nat Commun. 4: 1762). A Compact TALEN comprises an engineered, site-specific TAL-effector DNA-binding domain fused to the nuclease domain from the I-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. [0488] Engineered endonucleases based on the CRISPR/Cas9 system are also known in the art (Ran et al. (2013), Nat Protoc. 8:2281-2308; Mali et al. (2013), Nat Methods 10:957-63). The CRISPR gene-editing technology is composed of an endonuclease protein whose DNA-targeting specificity and cutting activity can be programmed by a short guide RNA or a duplex crRNA/TracrRNA. A CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically microbial Cas9; and (2) a short “guide RNA” or an RNA duplex comprising an 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).

[0489] 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.

[0490] 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.

[0491] 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.

[0492] In order to perform the gene editing technique, the nucleases, and in the case of the CRISPR/ Cas9 system, a gRNA, may need to be efficiently delivered to the cells of interest. Delivery methods such as physical, chemical, and viral methods are also know in the art (Mali (2013). Indian J. Hum. Genet. 19: 3-8.). In some instances, physical delivery methods can be selected from the methods but not limited to electroporation, microinjection, or use of ballistic particles. On the other hand, chemical delivery methods may 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. [0493] As an example, the endogenous TCR gene (e.g. , a TRAC locus or a TRBC locus) encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain can be inactivated by CRISPR/Cas9 system. The gRNA used to inactivate (e.g., disrupt) the TRAC locus can comprise a sequence of SEQ ID: 406. The gRNA used to disrupt the TRBC locus can comprise a sequence of SEQ ID: 197. As an example, the endogenous gene encoding a B2M subunit can be inactivated by CRISPR/Cas9 system. The gRNA used to inactivate (e.g., disrupt) the endogenous gene can comprise a sequence of SEQ ID NO: 196. [0494] CTCGACCAGCTTGACATCAC (SEQ ID NO: 406).

[0495] ACACTGGTGTGCCTGGCCAC (SEQ ID NO: 197). [0496] ACTCACGCTGGATAGCCTCC (SEQ ID NO: 196).

Methods of Treatment

[0497] Disclosed herein, in some embodiments, is a method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical compositions described herein. Further disclosed herein, in some embodiments, are methods of treating a disease or a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising (a) a cell produced according to the methods disclosed herein; and (b) a pharmaceutically acceptable carrier. In some embodiments, the disease or the condition is a cancer or a disease or a condition associated with expression of CD 19, B-cell maturation antigen (BCMA), mesothelin (MSLN), CD20, CD70, MUC16, Trop-2, Nectin-4, or GPC3. 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 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. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0498] Disclosed herein, in some embodiments, are methods of increasing the activity or persistence of a cell expressing a recombinant nucleic acid molecule comprising a sequence encoding the TFP disclosed herein, the method comprising expressing an interleukin- 15 (IL-15) polypeptide or a fragment thereof in the cell. Further disclosed herein, in some embodiments, are methods of increasing the activity or persistence of a cell expressing a recombinant nucleic acid molecule comprising a sequence encoding the TFP disclosed herein, the method comprising expressing an interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof in the cell. Disclosed herein, in some embodiments, are methods of increasing the activity or persistence of a cell expressing a recombinant nucleic acid molecule comprising a sequence encoding the TFP disclosed herein, the method comprising expressing a polypeptide or fusion protein that inhibits NK cell activity (e.g., a B2M-HLA-E and/or B2M-HLA-G) in the cell. Further disclosed herein, in some embodiments, are methods of increasing the activity or persistence of a cell expressing a recombinant nucleic acid molecule comprising a sequence encoding the TFP disclosed herein, the method comprising expressing a polypeptide or fusion protein that inhibits NK cell activity (e.g., a B2M-HLA-E and/or B2M-HLA-G) in the cell. In some embodiments, the cell is any one of cells described herein.

[0499] Disclosed herein, in some embodiments, are methods of reducing NK cell-mediated lysis of a cell described herein comprising or expressing a recombinant nucleic acid molecule comprising a sequence encoding the TFP disclosed herein, the method comprising expressing a polypeptide or fusion protein that inhibits NK cell activity (e.g., a B2M-HLA-E and/or B2M-HLA-G) in the cell.

[0500] Disclosed herein, in some embodiments, are methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical compositions disclosed herein. Further disclosed herein, in some embodiments, are methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a modified T cell produced according to the methods disclosed herein; and (b) a pharmaceutically acceptable carrier.

[0501] In some instances, the modified T cell is an autologous T cell. In some embodiments, the T cell is an allogeneic T cell. In some instances, less cytokines are released in the subject compared a subject administered an effective amount of an unmodified control T cell. In some instances, less cytokines are released in the subject compared a subject administered an effective amount of a modified T cell comprising the recombinant nucleic acid disclosed herein, or the vector disclosed herein.

[0502] In some instances, the method comprises administering the pharmaceutical composition in combination with an agent that increases the efficacy of the pharmaceutical composition. In some instances, the method comprises administering the pharmaceutical composition in combination with an agent that ameliorates one or more side effects associated with the pharmaceutical composition.

[0503] In some instances, the cancer is a solid cancer, a lymphoma or a leukemia. In some instances, the cancer is selected from the group consisting of renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney and stomach cancer.

[0504] In some embodiments, the present disclosure provides methods for treating cancer in a subject in need thereof, wherein the cancer has a low tumor antigen density. Thus, in some embodiments, the compositions and methods of the present disclosure provide a substantial improvement over CAR-T cell therapies and other engineered cell therapies that exhibit poor efficacy against tumors with low tumor antigen density.

[0505] The present disclosure includes a type of cellular therapy where T cells are genetically modified to express a TFP and an IL- 15 and/or IL-15Raand the modified 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, modified 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.

[0506] The present disclosure includes a type of cellular therapy where T cells are genetically modified to express a TFP and a fusion protein (e.g., B2M-HLA-E or B2M-HLA-G) and the modified T cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient and avoid host NK cell cytotoxicity. In some embodiments, the modified T cells further comprise an IL-15 and/or IL-15Ra as described herein.

[0507] The present disclosure also includes a type of cellular therapy where T cells are modified, e.g. , by in vitro transcribed RNA, to transiently express a TFP and an IL- 15 and/or IL-15Ra and/or NK cell inhibiting agent (e.g., B2M-HLA-E fusion protein or B2M-HLA-G fusion protein) as described herein, and the modified 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.

[0508] Without wishing to be bound by any particular theory, the anti-tumor immunity response elicited by the modified T cells may be an active or a passive immune response, or alternatively may be due to a direct vs indirect immune response.

[0509] In one aspect, the human modified T cells of the 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.

[0510] 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 and an IL-15 and/or IL-15Ra and/or NK cell inhibiting agent to the cells or iii) cryopreservation of the cells. [0511] 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 disclosed herein. The modified T cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the modified cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.

[0512] The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described 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-L, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.

[0513] 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.

[0514] 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.

[0515] The 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 other cytokines or cell populations.

Combination Therapies

[0516] A modified T 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.

[0517] In some embodiments, the “at least one additional therapeutic agent” includes a modified T 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.

[0518] A modified T 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 modified T cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.

[0519] In further aspects, a modified T 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, cytoxin, 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.

[0520] In one embodiment, the subject can be administered an agent which reduces or ameliorates a side effect associated with the administration of a modified T cell. Side effects associated with the administration of a modified T 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 disclosed herein can comprise administering a modified T cell described herein to a subject and further administering an agent to manage elevated levels of a soluble factor resulting from treatment with a modified T 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 entanercept. An example of an IL-6 inhibitor is tocilizumab (toe). [0521] In one embodiment, the subject can be administered 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., Programmed Death 1 (PD-1), can, in some embodiments, decrease the ability of a modified T cell to mount an immune effector response. Examples of inhibitory molecules include PD-1, 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 modified T 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 modified T 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-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX-101 and marketed as Yervoy®; Bristol-Myers Squibb; tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP -675, 206)). In an embodiment, the agent is an antibody or antibody fragment that binds to TIM3. In an embodiment, the agent is an antibody or antibody fragment that binds to LAG3.

[0522] In some embodiments, the agent which enhances the activity of a modified T cell can be, e.g., a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule, or fragment thereof, and the second domain is a polypeptide that is associated with a positive signal, e.g. , a polypeptide comprising an intracellular signaling domain as described herein. In some embodiments, the polypeptide that is associated with a positive signal can include a costimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27 and/or ICOS, and/or a primary signaling domain, e.g., of CD3 zeta, e.g., described herein. In one embodiment, the fusion protein is expressed by the same cell that expressed the TFP. In another embodiment, the fusion protein is expressed by a cell, e.g., a T cell that does not express an anti-TAA TFP.

EXAMPLES

Example 1: TFP constructs

[0523] Inactivation of the TRAC or TRBC genes in Jurkat cells was done by electroporation of SpCas9 ribonucleoproteins (RNPs) directed against TRAC or TRBC genes. Cells were maintained at 0.2xl0⁶ cells per mL in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and 300mg/L L-Glutamine until electroporation. SpCas9 ribonucleoproteins targeting TRA or TRB genes were prepared by annealing crRNA targeting either TRAC (TRAC2-4598) or TRBC (TRBC-44345) with tracrRNA at a molecular ratio of 1: 1. Annealed duplexes were mixed with SpCas9 protein at a molecular ratio of 1.5: 1. 0.61 pM of RNPs were mixed with 2.5xl0⁶ T cells and electroporated according to the manufacturer’s protocol for the Neon Transfection System (Thermo Fisher Scientific). Electroporation was set at 1600V, 10ms, 3 pulses. After pulse the cells were immediately transferred to warm medium and incubated at 37°C for three days.

[0524] Editing efficacy was assessed by observing loss of surface expression of TCRaP and CD3a via flow cytometry. Edited Jurkat cells were purified via Magnetic -Activated Cell Sorting (MACS, Miltenyi Biotec) cell separation system. Edited Jurkat cells were negatively selected against anti -TCRaP (clone: IP27) (eBioscience #17-9986-42) antibody and anti-CD3a (clone:SK7) antibody (eBioscience #25-0036-42). Cells expressing TCRaP or CD3s at their surface were immobilized to MACS MS (Cat. #130-041-301) or LS (Cat. #130-041-306) columns, while edited Jurkat cells, negative for both TCRaP or CD3s were collected in the column flow through and maintained in culture at 0.4xl0⁶ cells/mL in the medium specified above. TCRa and TCRp knock out cells are herein called TRA-/- or TRB-/- Jurkat cells.

Transduction of Jurkat cells

[0525] TFP transgenes were introduced in Jurkat cells using lentiviruses. Jurkat cells were incubated with virus at a multiplicity of infection (MOI) of five. Medium was replaced twenty-four-hours post incubation. Transduction efficacy and TFP expression was assessed with flow cytometry using a ligand specific to the TFP binder of interest and/or surface expression of TCRaP or CD3s. TRAC-/- and TRBC-/- Jurkat cells were transduced with TCRyS TFPs and restoration of surface TCR was indicated by highly positive CD3s staining. Transduction ofT cells

[0526] TFP transgenes were introduced into T cells using lentiviruses. T cells were mixed together with viruses at a multiplicity of infection (MOI) of five plus lOOng/mL of LentiBOOST™ (Sirion Biotech). Transduction efficacy and TFP expression was assessed with flow cytometry using a ligand specific to the TFP binder of interest and/or surface expression of TCRaP or CD3s.

Description of Transgenes [0527] In a/p T cells inactivation of TRAC or TRBC blocks the translocation to the cell surface of all TCR subunits. TCRa or TCR cannot pair with TCRy or TCR5. Consequently, an exogenous TRGC and TRDC transgenes or TRAC and TRBC transgenes are expressed in TRAC ^/_ or TRBC ^/_ cells to restore a functional TFP T cell.

Expression of human TCRy/STFP

[0528] TCRa negative cells still express TCR and, reciprocally, TCRa is expressed in TCR negative cells; However, TCRa or TCR cannot pair with TCRy or TCR5. Therefore, TCRy TFP and TCR5 TFPs were expressed together in TRAC ^/_ cells or in TRB ^/_ cells. Multiple formats of TCRy/5 TFPs were tested in TCR negative cells to determine the optimal construction to restore translocation of the entire TCR complex. In one embodiment, TCRy/5 TFPs were generated by assembling the constant domains of TCRy or/and TCR5 with an antigen binder (e.g., scFv or sdAb). In another embodiment, TCRy/5 constant domains are expressed together with a CD3s TFP TRGC1 and TRDC residues are numerated according to the the sequences provided herein and according to international ImMunoGeneTics information system (IMGT).

[0529] In some cases, an anti-MSLN 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. Source of TCR Subunits

[0530] A 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 can 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. Q6ISU1. The murine TCR alpha chain canonical sequence is Uniprot Accession No. A0A075B662. The human TCR beta chain constant region canonical sequence is Uniprot Accession No. P01850. The murine TCR beta chain constant region canonical sequence is Uniprot Accession No. P01852.

[0531] The human CD3 -epsilon polypeptide canonical sequence is: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIG GDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATI VIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYS GLNQRRI (SEQ ID NO: 124).

[0532] The mature human CD3 -epsilon polypeptide sequence is: DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELE QSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKA KAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:258). [0533] The signal peptide of human CD3a is: MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO: 125).

[0534] The extracellular domain of human CD3a is:

DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELE QSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 126).

[0535] The transmembrane domain of human CD3s is: VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID NO: 127).

[0536] The intracellular domain of human CD3s is:

KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 128).

[0537] The human CD3 -gamma polypeptide canonical sequence is:

MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTE

DKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGV YFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 129).

[0538] The mature human CD3 -gamma polypeptide sequence is:

QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQC

KGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLP NDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 130).

[0539] The signal peptide of human CD3y is: MEQGKGLAVLILAIILLQGTLA (SEQ ID NO: 131).

[0540] The extracellular domain of human CD3y is:

QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQC

KGSQNKSKPLQVYYRMCQNCIELNAATIS (SEQ ID NO: 132).

[0541] The transmembrane domain of human CD3y is: GFLFAEIVSIFVLAVGVYFIA (SEQ ID NO: 133).

[0542] The intracellular domain of human CD3y is:

GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 134).

[0543] The human CD3 -delta polypeptide canonical sequence is:

MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGI

YRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAA DTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKS (SEQ ID NO: 135).

[0544] The mature human CD3 -delta polypeptide sequence is:

FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHY

RMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDD AQYSHLGGNWARNKS (SEQ ID NO: 136).

[0545] The signal peptide of human CD35 is: MEHSTFLSGLVLATLLSQVSP (SEQ ID NO: 137).

[0546] The extracellular domain of human CD35 is:

FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHY RMCQSCVELDPATVA (SEQ ID NO: 138).

[0547] The transmembrane domain of human CD35 is: GUVTDVIATLLLALGVFCFA (SEQ ID NO: 139). [0548] The intracellular domain of human CD35 is:

GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO: 140).

[0549] The human CD3-zeta polypeptide canonical sequence is:

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 141).

[0550] The human TCR alpha chain constant region canonical sequence is:

IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSN

KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLR LWSS (SEQ ID NO: 142).

[0551] The human TCR alpha chain human IgC sequence is:

IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSN

KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS (SEQ ID NO: 143)

[0552] The transmembrane domain of the human TCR alpha chain is:

VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 144).

[0553] The intracellular domain of the human TCR alpha chain is: SS (SEQ ID NO: 145)

[0554] The murine TCR alpha chain constant (mTRAC) region canonical sequence is:

XIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSN

QTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRL WSS (SEQ ID NO: 146).

[0555] The transmembrane domain of the murine TCR alpha chain is: MGLRILLLKVAGFNLLMTLRLW (SEQ ID NO: 147).

[0556] The intracellular domain of the murine TCR alpha chain is: SS (SEQ ID NO: 145)

[0557] The human TCR beta chain constant region (mTRBC) canonical sequence is:

EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQP

ALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF TSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 148).

[0558] The human TCR beta chain human IgC sequence is:

EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQP ALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF TSVSYQQGVLSATILYE (SEQ ID NO: 149)

[0559] The transmembrane domain of the human TCR beta chain is: ILLGKATLYAVLVSALVLMAM (SEQ ID NO: 150).

[0560] The intracellular domain of the human TCR beta chain is: VKRKDF (SEQ ID NO: 151)

[0561] The murine TCR beta chain constant region canonical sequence is:

EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKES NYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSAS YQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 152).

[0562] The transmembrane domain of the murine TCR beta chain is: ILYEILLGKATLYAVLVSTLVVMAMVK (SEQ ID NO: 153).

[0563] The intracellular domain of the murine TCR beta chain is: KRKNS (SEQ ID NO: 154)

[0564] The human TCR gamma chain constant region canonical sequence is: DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDT YMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNT SAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS (SEQ ID NO:21).

[0565] The human TCR gamma human IgC sequence is: DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDT YMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNT SA (SEQ ID NO: 155)

[0566] The transmembrane domain of the human TCR gamma chain is: YYMYLLLLLKSVVYFAIITCCLL (SEQ ID NO: 156).

[0567] The intracellular domain of the human TCR gamma chain is: RRTAFCCNGEKS (SEQ ID NO: 157) [0568] The human TCR delta chain C region canonical sequence is: SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSV TCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRML FAKTVAVNFLLTAKLFFL (SEQ ID NO:243).

[0569] The human TCR delta human IgC sequence is: SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSV TCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTV (SEQ ID NO: 265)

[0570] The transmembrane domain of the human TCR delta chain is:

LGLRMLFAKTVAVNFLLTAKLFF (SEQ ID NO: 158).

[0571] The intracellular domain of the human TCR delta chain is: L

IL-15 peptides

[0572] In some embodiments, TFP constructs are in a vector that further contains a sequence encoding an IL- 15 peptide or an IL15-Ra peptide. The IL- 15 may be encoded in the same open reading frame and separated by a self-cleaving peptide (e.g., a P2A or a T2A self-cleaving peptide). In some embodiments, the IL-15 peptide comprises a secreted IL-15. The secreted IL-15 can have the sequence of SEQ ID NO: 375. In some embodiments, the IL-15 peptide is an IL-15-IL15Ra fusion. In some embodiments, IL-15Ra comprises the sequence of SEQ ID NO: 383 or SEQ ID NO: 386. In some embodiments, the IL-15-IL15Ra fusion comprises a linker followed by a sushi domain linking IL-15 and IL-15Ra. In some embodiments, the IL-15-IL15Ra fusion comprises the sequence of SEQ ID NO: 389. [0573] IL- 15 -IL 15 Ra fusion: GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVI SLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSG GGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVL NKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAI VPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLL CGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL. (SEQ ID NO: 389) TFP Expression Vectors

[0574] Expression vectors are provided that include: a promoter (e.g., an EFla promoter), a signal sequence to enable secretion, a polyadenylation signal and transcription terminator (Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes (e.g., SV40 origin and ColEl or others known in the art) and elements to allow selection (ampicillin resistance gene and zeocin marker). [0575] Preferably, the TFP-encoding nucleic acid construct with or without an IL-15/IL15Ra peptide and with or without an HLA-E or HLA-G poly peptide or fusion protein is 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 (e.g., degranulation).

Example 2: Generation and characterization of double knockout allogeneic T cell receptor fusion construct modified T cells

[0576] It has been described previously the generation of allogeneic T cells comprising functional disruption (e.g., knockdown or knockout) of one or more endogenous T cell receptor subunit genes (e.g., TCR alpha and/or beta subunits) with concomitant expression of exogenous TFPs for the treatment of cancer (see International Publication W02021050948, the contents of which are herein incorporated by reference in their entirety). Here it was tested whether introduction of a second functional disruption in the modified T cells would result in continued specific tumor targeting activity and improved host response.

[0577] A recombinant nucleic acid construct encoding mesothelin (MSLN) targeting allogeneic TFP MHld MHlg (SEQ ID NO: 361) was cloned into a pLKaUS vector and transduced via lentivirus into activated T cells harvested from healthy donors. The construct included sequence regions encoding two separate MSLN binding regions (SEQ ID NO: 69), operatively linked to T cell receptor constant domain delta (TRDC; SEQ ID NO: 243) and T cell receptor constant domain gamma (TRGC; SEQ ID NO: 21), respectively, separated by GSG, furin (SEQ ID NO: 363), and self-cleaving peptide (P2A; SEQ ID NO: 365) sequence regions. The construct components and related sequences as read 5’ to 3’ or N-terminus to C- terminus are outlined in Table 5.

[0578] Three days later, the transduced T cells underwent genome editing for knockout of the T cell receptor alpha (TRAC) and/or beta-2 -microglobulin (B2M) genes using CRISPR/Cas9 systems. It was hypothesized that double knock-out of endogenous TCRa and B2M in allogeneic T cells expressing MSLN targeting TFPs would successfully prevent graft versus host disease and host rejection while maintaining MSLN specific cytotoxicity. TRAC and B2M genes were targeted using guide sequences as given by: TRAC (CTCGACCAGCTTGACATCAC; SEQ ID NO: 406) and B2M (ACTCACGCTGGATAGCCTCC; SEQ ID NO: 196), respectively. TRAC/Cas9 (1.8 pM) and B2M/Cas9 (1.8 pM) ribonucleoprotein (RNP) complex formation was completed separately, with RNPs combined (3.6 pM) only at electroporation. For electroporation, 50 pL of electroporation buffer/RNP in OC- 100x2 was provided to approximately 2E6 T cells. Modified T cells demonstrating successful hTCRaP knockout were purified, expanded, and cryopreserved for later study. A schematic of a non-limiting, exemplary, double knockout TFP expressing modified T cell is shown in FIG. 1. In some embodiments, the double knockout TFP expressing modified cell further expresses a B2M-HLA-E or B2M-HLA-G, for example, a mutated B2M-HLA-E fusion protein. [0579] To characterize knockout efficiency and TFP expression and function in these modified T cells, the following 9 experimental conditions were generated (i) non-transduced (NT), no knockout (KO), (ii) NT, TRAC KO, (iii) NT, B2M KO, (iv) NT, TRAC.B2M double knockout (DKO), (v) MHle (SEQ ID NO: 195), No KO, (vi) MHld MHlg (SEQ ID NO: 361), No KO, (vii) MHld MHlg, TRAC KO, (viii) MHld MHlg, B2M KO, and (ix) MHld MHlg, TRAC.B2M DKO. Using flow cytometry methods, individual TCR and B2M knockout efficiency was quantified across T cells collected from three separate donors (biological triplicate) and demonstrated that multiplexing TRAC and B2M/Cas9 resulted in high TCR knockout efficiency (>90% TCR-) and high B2M knockout efficiency (>70% B2M-). Each of the TRAC KO conditions showed substantially similar knockout efficiency regardless of single or double knockout and/or combination with TFP transduction. Evaluation of the conditions wherein B2M was knocked out yielded the same patterns, though with slightly less overall knockout efficiency to that seen with TRAC KO across all groups.

Quantification of the efficiency of the TRAC/B2M double knockout also showed high efficiency (around 65% DKO) after multiplexed TRAC/B2M Cas9 mediated genome editing.

[0580] Further characterization of the B2M KO modified T cells by additional detection of HLA-A/B/C showed a consistent presence (5/5) of a population HLA-Class I-/B2M+ cells, suggestive of HLA knockout greater than B2M knockout alone. These data are shown in FIG. 2 and indicated that B2M KO was not equivalent to HLA-Class I KO. HLA-A/B/C surface expression was then calculated using flow cytometry and showed high efficiency (-80%) HLA-Class I knockout after multiplexed TRAC/B2M Cas9 mediated genome editing. Each of the B2M KO conditions showed substantially similar HLA Class I knockout efficiency regardless of single or double knockout and/or combination with TFP transduction. These data are shown in FIG. 3. Double knockout as measured by detection of HLA-A/B/C and TCRaP by flow cytometry again showed high efficiency with approximately 75% DKO determined for both the NT and transduced DKO conditions. The editing efficiency of the B2M/Cas9 RNP was also assessed by CRISPR Seq with quantification of percent indel efficiency. In both the NT, B2M KO and NT, TRAC/B2M DKO conditions, indel efficiency was calculated to be about 90%, indicating high editing efficiency. When knockdown efficiency was calculated subsequent to hTCRaP purification on Day 7, TCR knockout efficiency climbed to 100% while no significant change in percent knockout efficiency was seen for the B2M KO or DKO conditions.

[0581] Modified T cells were also assessed for MSLN targeting TFP transduction efficiency by detecting the surface expression of the VHH MSLN binding domains using flow cytometry (FIG. 4). Cells transduced with MHle or MHld MHlg showed high transduction efficiency regardless of knockout status (no KO, single KO or DKO) indicating that TRAC/B2M DKO did not substantively impact TFP transduction and VHH expression.

[0582] Quantification of T cell phenotype based on percent expression of CD4 and CD8 in the modified T cells showed substantially similar CD4/CD8 phenotype across conditions. These data supported the concept that TRAC/B2M DKO modified T cells were not negatively impacted by the second knockout and showed similar T cell phenotype to single knockout and no knockout modified T cells. Memory phenotype of the CD4+ and CD8+ cells was also evaluated. In these studies, TRAC/B2M DKO did not impact memory phenotype when compared to single knockout and no knockout conditions, regardless of transduction. CD4+ MHld MHlg DKO modified T cells were determined to be primarily effector memory cells (CD45RA- /CCR7-) while CD8+ MHld MHlg DKO modified T cells were determined to be primarily naive cells (CD45RA+/CCR7+).

[0583] T cell exhaustion for the modified T cells was assessed by quantification of percent expression of TIGIT, LAG-3, TIM-3, PD-1 and TIM-3/PD-1. Across all T cell conditions and all three donors, TIM-3 expression was high. In this assay, the DKO modified T cells were not significantly different from modified T cells in other conditions (e.g., NT or transduced, no knockdown or single knockdown).

[0584] Activation kinetics when stimulated with MSTO-MSLN+, C30 MTLN-, or no stimulus were determined based on CD69, CD25, HLA-Dr, and CD70 percent surface expression overtime (Day 0 to Day 4). In each condition, the DKO modified T cells performed much like the modified T cells of the single KO and/or no KO comparator conditions. Cell recovery post freeze/thaw was also shown to be substantially similar across conditions and not specifically impacted in the DKO conditions.

[0585] The ability of the modified T cells of each condition to expand over the 10-day time-period was also evaluated. Transduced TRAC/B2M DKO cells had slightly reduced expansion when compared to single KO or NT DKO modified T cells.

[0586] In an in vitro cytotoxicity assay against MSLN expressing target cells, MHld_MHlg TRAC/B2M DKO modified T cells demonstrated substantially similar lysing ability to TFP transduced single KO or no KO modified T cells, as shown in FIG. 5.

[0587] These studies demonstrated successful generation of double knockout allogeneic TFP expressing modified T cells, which retained their phenotype and cytotoxicity against MSLN expressing target cells.

Example 3: Evaluation of membrane-tethered IL-15/IL-15 receptor fusion proteins as an enhancement for allogeneic TFP expressing modified T cells [0588] To test an alternative enhancement to allogeneic T cells comprising functional disruption (e.g., knockdown or knockout) of one or more endogenous T cell receptor subunit genes (e.g., TCR alpha and/or beta subunits) with concomitant expression of exogenous TFPs for the treatment of cancer (see International Publication W02021050948, the contents of which are herein incorporated by reference in their entirety), coexpression of a membrane-tethered IL-15/IL-15 receptor fusion protein was evaluated.

[0589] Exemplary schematics of the constructs tested in these experiments are shown in FIG. 6 and the sequences and components thereof are outlined in Table 5.

[0590] T cells collected from healthy donors were thawed and activated (Day 0), then transduced (Day 1) with lentiviral vectors carrying recombinant nucleic acid sequences encoding one of the four constructs shown in FIG. 6 and outlined in Table 5. In the first condition tested, the construct (MHld MHlg; SEQ ID NO: 361 included sequence regions encoding two separate MSLN binding regions (SEQ ID NO: 69), operatively linked to T cell receptor constant domain delta (TRDC; SEQ ID NO: 243) and T cell receptor constant domain gamma (TRGC; SEQ ID NO: 21), respectively, separated by GSG, furin (SEQ ID NO: 363), and self-cleaving peptide P2A (SEQ ID NO: 365) sequence regions. In the second condition tested (MHld MHlg + IL15fiis; SEQ ID NO: 366), an IL-15/IL-15Ra fusion construct (SEQ ID NO: 371) linked by an SG3(SG4)3SG3SLE linker was together operatively linked to the 3’ end of the MHld_MHlg construct sequence via a T2A cleavage sequence (SEQ ID NO: 23). A linker was introduced between the MSLN binding sequences and TCR constant domains for the third tested condition (MHld MHlg + linker; SEQ ID NO: 367) and in the fourth (MHld MHlg + linker + IL15fus; SEQ ID NO: 368), an IL15/IL-15Ra fusion construct was tested as well. An MSLN targeting autologous TFP (MHle; SEQ ID NO: 195) was used as an additional comparison condition.

[0591] On Day 4, in each of the modified T cell conditions tested, a T cell receptor alpha (TRAC) gene knockout was introduced using a CRISPR/Cas9 system. On Day 7, cells were purified and modified T cells showing effective knockout of TRAC (hTCRa[3-) were purified and assessed by flow cytometry. The T cells were then given time to expand and harvested on Day 10 for post thaw characterization.

[0592] Flow cytometry analysis of transduction efficiency in modified T cells showed that each of the TFP and IL 15 fusion constructs was successfully expressed, but that those conditions in which an IL 15 fusion protein was also introduced showed a slightly decreased transduction efficiency. Characterization of the transduced modified T cells showed a higher CD4+ population in those cells expressing the IL15fiision protein. Memory phenotype was also determined.

[0593] A standard in vitro cytotoxicity assay was conducted against MSTO-MSLN tumor cells. Each of the tested conditions, including the MHle control showed substantially similar levels of cytotoxicity as determined by quantification of percent tumor cell lysis after 24hrs of co-culture. These data are shown in FIG. 7. Cytokine secretion was also assessed and each of the allogeneic modified T cells showed roughly equivalent cytokine release as that seen with MHld_MHlg against MSTO-MSLN cells as measured by IL-2, TNFa, GM-CSF, IFNy. [0594] In an in vivo assay, the overall efficacy data proved to be similar to that of the in vitro assay, but each of the allogeneic conditions tested performed better than the autologous MHle control in keeping the tumor from reemerged as determined by tumor volume measurement over time. These data are shown in FIG. 8. [0595] Ex-vivo analysis of blood, tumor, liver and spleen tissues at Day 19 and Day 38 showed increased hCD45+ cell counts in MHld MHlg and MHld MHlg + IL15fus conditions as compared to MHle in blood, as shown in FIG. 9, as well as in tumor, liver and spleen (data not shown). These data suggested an enhanced expansion in vivo of the allogeneic modified T cells. At Day 38, only the allogeneic modified T cell also having an IL15fus protein showed persistence in blood.

[0596] Together these data suggested that an IL- 15 fusion protein may be useful in enhancing the anti- tumoral effects of allogeneic modified T cells.

Example 4: Generation and characterization of double knockout allogeneic T cell receptor fusion construct modified T cells with a membrane tethered IL-15/IL-15 receptor fusion protein enhancement [0597] The allogeneic modified T cell enhancements described in Examples 2 and 3 (e.g., TRAC/B2M double knockout) and IL-15/IL-15 receptor fusion protein enhancement will be tested within the same T cell to evaluate whether these enhancements may work synergistically in terms of expression, function and efficacy. T cells collected from healthy donors will be activated and then transduced with lentiviral vectors carrying recombinant nucleic acids encoding one or more TFP construct(s) and, in some cases, additionally a membrane tethered IL-15/IL-15 receptor fusion protein (IL15fus). Transduced T cells will be subjected to CRISPR/Cas9 genome editing for knockout of TRAC and/or B2M genes. Modified T cells will then be assessed by flow cytometry for characterization of knockout efficiency and TFP and/or IL15fus surface expression. T cell and memory phenotypes can also be quantified. Other assays to evaluate T cell exhaustion, post thaw activation kinetics, expansion and more can be conducted. Modified T cells will be tested in vitro and in vivo for functionality, tumor targeting, efficiency, specificity, expansion and persistence.

Example 5: Engineering allogeneic mesothelin targeting T cell receptor fusion construct modified T cells

[0598] The development of autologous mesothelin (MSLN) targeting T cell receptor fusion construct (TRuC™ or TFP) T cells (TC-210) has been previously described (see e.g., International Application publication WO2018067993, the contents of which are herein incorporated by reference in their entirety). Here “off-the-shelf’ allogeneic MSLN targeting TRuC T cells were engineered and tested against the autologous TC-210 in a head-to-head comparison.

[0599] As previously described, TC-210 (or MHle; SEQ ID NO: 195) was engineered to include a single domain antibody (sdAb) targeting MSLN, “MH1” (SEQ ID NO: 69), fused to the extracellular domain of CD3 epsilon. Whilst not wishing to be bound by theory, T cells transduced to express TC-210 retain endogenous ot/ TCR subunits and may therefore be vulnerable to reaction with “non-self ’ molecules and Graft versus Host Disease (GvHD). To reduce the risk of GvHD, allogeneic MSLN targeting TRuC T cells were generated. First, the T cell receptor Alpha Constant (TRAC) gene was targeted and knocked-out in healthy donor T cells using a CRISPR/Cas9 system having guide sequence CTCGACCAGCTTGACATCAC (SEQ ID NO: 406), thereby eliminating the surface expression of the native TCR and related alloreactivity. These modified T cells (i.e., having a functional disruption of the TRAC gene) were then transduced with lentiviral vectors encoding MHld MHlg (or MHlyS; SEQ ID NO: 361) to enable the assembly of non- alloreactive TCR complexes. The MHld MHlg transgene was designed to incorporate two of the MH1 anti- MSLN sdAb binders used in TC-210 fused to TCR gamma and TCR delta, respectively, as shown in FIG. 10. [0600] T cells transduced with MHld MHlg or TC-210 and non-transduced control T cells were assessed by flow cytometry for the surface expression of the MH1 MSLN binder as determined by staining with an anti- VHH antibody. Exemplary flow cytometry plots are shown in FIG. HA. The data demonstrated that the transduction efficiency was similar when introducing the MHld MHlg or TC-210 TFP to T cells. Transduced T cells were further characterized as CD4+/CD8+ and by memory phenotype based on the detection of CD45RA and CCR7 using flow cytometry. FIG. 11B shows stacked bar plots for the memory phenotype populations (Naive = CD45RA+/CCR7+, central Memory (Tcm) = CD45RA-/CCR7+, effector memory (Tem) = CD45RA-/CCR7-, and terminally differentiated effector memory (Temra) = CD45RA+/CCR7-) in CD4+ and CD8+ transduced T cells across 5 donors.

[0601] To assess the in-vitro efficacy of the transduced T cells, a luciferase-based cytotoxicity assay was conducted. MHld MHlg and TC-210 transduced T cells and non-transduced control cells were co-cultured with C30-luc (MSLN negative) and MSTO-MSLN-Luc (MSLN expressing) cells at effector (TRuC T cells) to target (C30-luc or MSTO-MSLN-Luc) (E:T) ratios of 9: 1, 3: 1, 1: 1 and 1:3 for 24 hours. After the 24hr coculture, the luciferase readout of the remaining cells was used to inversely calculate percent tumor lysis. The data for the co-culture with MSTO-MSLN-luc target cells are shown in FIG. 11C and demonstrated that both MHld_MHlg and TC-210 transduced T cells were effective in killing MSLN expressing target cells.

[0602] As another readout of in-vitro efficacy of transduced T cells, cytokine release was assessed by quantifying the presence of IL-2 and TNFa in the supernatant of the 24hr co-culture noted above. These data are shown in FIG. HD and suggested that MHld MHlg transduced T cells promoted more IL-2 and TNFa release after 24hr of co-culture at E:T ratio of 9: 1 when compared to TC-210 transduced T cells. The cytotoxicity and cytokine release assays were conducted using two donors.

[0603] The efficacy of MHld MHlg and TC-210 TRuC T cells was further tested in-vivo in a mouse model. NSG mice were engrafted with a subcutaneous dose of le6 MSTO-MLSN-Luc cells mixed with Matrigel in the right flank. Tumor size was followed overtime and once tumors reached 250-350mm³ in size (day 21 post tumor engraftment), 2e6 MHld MHlg or TC-210 TRuC T cells were injected intravenously. Tumor size was quantified every 3-4 days and plotted over time. Tumor growth curves for 5 distinct studies (and donors) are shown in FIG. 12. The data indicated that animals treated with allogeneic MHld MHlg TRuC T cells had prolonged tumor clearance as compared to animals treated with autologous TC-210 TRuC T cells.

[0604] To assess in vivo TRuC T cell expansion and proliferation, tissues (blood, spleen, liver) were harvested on Day 19 post TRuC T cell injection and analyzed. Graphs showing the normalized counts of TRuC T cells as determined by human CD45 detection (hCD45+) and subsequent VHH detection (TRuC+) are given in FIG. 13. Results were combined across 3 donors (n=9). These data showed that allogeneic MHld_MHlg TRuC T cells were detected longer and had enhanced persistence in vivo when compared to the autologous TC-210 TRuC T cells.

[0605] Since the MSTO-MSLN-luc cells used for engraftment in the previous study expressed high levels of MSLN, a challenge study was conducted using a cell line with lower antigen (MSLN) density (SUIT2-luc cells). NSG mice were engrafted with SUIT2-luc cells, then 8-10 days later received TRuC T cell injection. In this study, a membrane -tethered IL-15/IL-15 receptor fusion (IL15fus) enhancement was tested in combination with the MHld MHlg (SEQ ID NO: 366) to determine if TRuC T cell persistence could be further improved. Tumor volumes were measured every 3-4 days over time and plotted as shown in FIG. 14A. Allogeneic TRuC T cells with or without co-expression of the IL 15 fusion protein successfully cleared tumors in this low MSLN expressing model system, while TC-210 did not exhibit tumor clearance in this model. Preliminary comparison of the MHld MHlg alone versus in combination with the IL15 fusion protein suggested a trend toward improved efficacy of the MHld MHlg enhanced with the IL15fiision protein. The group averages are shown in FIG. 14B (left panel). When assessed by individual animal, three of five animals in the MHld MHlg + IL15fus group resisted tumor regrowth longer than animals receiving MHld MHlg alone, with 1 animal maintaining tumor clearance (tumor volume of 0 mm³) throughout the duration of the study (FIG. 14B).

[0606] The allogeneic MHld MHlg TRuC T cells tested in these experiments were designed to incorporate functional disruption of a single gene (TRAC). To test whether host rejection could be further reduced while retaining function, a second gene knock-out (double knock out; DKO) was introduced and compared to single TRAC knock-out (KO) TRuC T cells and no knock-out controls. Beta-2 microglobulin (B2M) was selected as the second target for knock-out, to eliminate MHC Class 1 surface expression. A multiplexed CRISPR/Cas9 system was used to knock-out TRAC using guide sequence CTCGACCAGCTTGACATCAC (SEQ ID NO: 406) and/or B2M using guide sequence ACTCACGCTGGATAGCCTCC (SEQ ID NO: 196). Modified T cells (TRAC KO and TRAC/B2M DKO) were then transduced to express MHld MHlg (SEQ ID NO: 361). No knock-out TC-210 transduced T cells were used as controls.

[0607] Subsequent to gene editing, transduction, and expansion, cells were collected and assessed for surface expression of HLA A/B/C and hTCRaP, using flow cytometry. Exemplary plots are shown in FIG. 15A and demonstrate that TCRaP and HLA-1 were successfully eliminated from the surface of TRAC/B2M DKO T cells. Modified T cells were further characterized as CD4+/CD8+ and classified by memory phenotype based on the detection of CD45RA and CCR7 using flow cytometry. FIG. 15B shows stacked bar plots for the memory phenotype populations (Naive = CD45RA+/CCR7+, central Memory (Tcm) = CD45RA-/CCR7+, effector memory (Tem) = CD45RA-/CCR7-, and terminally differentiated effector memory (Temra) = CD45RA+/CCR7-) in CD4+ and CD8+ transduced T cells across 5 donors. These data indicated that CD4+/CD8+ and memory phenotypes were substantially similar across all three groups, demonstrating that introduction of a second gene knock-out did not substantially alter the T cell memory phenotype. [0608] The in vitro function of the modified T cells was tested using a luciferase-based cytotoxicity assay (described previously), wherein T cells were co-cultured for 24hr with MSTO-MSLN-luc cells at variable E:T ratios (9: 1, 3: 1, 1: 1 and 1:3), then the luciferase read-out was used to inversely quantify the tumor cell lysis. The resultant data are shown in FIG. 15C. Cell killing ability was substantially similar across groups, again suggesting that the double knock-out did not negatively impact the in vitro function of the allogeneic MHld MHlg TRuC T cells.

[0609] Activation status of the modified T cells was evaluated using an assay in which MHld MHlg TRAC KO, MHld MHlg TRAC/B2M DKO, no knock-out TC-210, and non-transduced control cells were cocultured with MSTO-MSLN cells. At 24, 48, and 96 hours post stimulation, the cells were stained for activation markers CD69, CD25, CD70 and HLA-DR and the percent of TrucC+ cells that were positive for each marker was quantified. Transduced T cells, regardless of knock-out status, showed substantially similar activation marker profiles as shown in FIG. 15D, again suggesting that single or double gene knock-out was endured without loss of in vitro functionality in TRuC T cells.

[0610] In vivo efficacy of single and double knock-out allogeneic TRuC T cells was tested in a mouse model. NSG mice were engrafted with MSTO-MSLN-luc cells and 21 days later provided with MHld_MHlg TRAC KO, MHld MHlg TRAC/B2M DKO, or non-transduced control cells. A vehicle control was also included. Tumor volumes were measured every 3-4 days. Both MHld MHlg TRAC KO and MHld MHlg TRAC/B2M DKO TRuC T cells were able to successfully clear tumor cells with roughly equivalent potency (FIG. 15E). DKO allogeneic TRuC T cells were able to maintain potency similar to that seen with the single TRAC KO.

[0611] Taken together, these studies indicated that allogeneic TRuC T cells having a TRAC KO and expressing MHld_MHlg had similar in vitro characteristics and efficacy against MSLN expressing tumors when compared to their autologous TC-210 counterparts. Allogeneic TRuC T cells were able to upregulate activation markers, secrete robust cytokines, and lyse tumor cells in an antigen-specific manner without alloreactivity. In vivo, the allogeneic TRuC T cells showed prolonged persistence and improved sensitivity and efficacy against low density MSLN expressing SUIT2 tumor cells. When a second gene knock-out was introduced to the allogeneic TRuC T cells for an improved alloreactivity profile, the cells retained full potency.

Example 6. Evaluation of fusion protein constructs containing B2M-HLA-E

[0612] A study was conducted to evaluate the feasibility of incorporating NK cell inhibitory signals in double knock out allogeneic TFP expressing modified T cells, in order to reduce or eliminate potential NK cell activity against the modified T cells. Several constructs were designed and tested to determine if HLA-E expression could be achieved in B2M knockout primary T cells. An exemplary construct is shown in FIG. 16. The exemplary construct comprised a B2M signal sequence, HLA-G binding peptide, G4S linker, mutated B2M, a second G4S linker, and HLA-E*01:03. Other fusion proteins including those employing a GMCSFR signal peptide rather than a B2M signal sequence, and/or without the HLA-G binding peptide, were also generated and transduced into cells for determination of and HLA-E expression. The structure of a fusion protein with or without the HLA-G binding peptide is shown in FIG. 16. In addition, the B2M portion of the fusion protein incorporated a mutation at the sgRNA binding site and PAM site to prevent cleavage by Cas9 during the generation of a B2M KO T cell. The construct with the B2M signal sequence and HLA-G binding peptide resulted in the highest % HLA-E expression and greatest mean MFI of the constructs tested.

[0613] B2M knockout primary T cells were transduced with the fusion protein construct shown in FIG. 16. As shown in FIG. 17, the transduced cells exhibited high expression of HLA-E. The expression of HLA-E protected B2M KO primary T cells from NK cell mediated cytotoxicity, as shown in FIG. 18. Similarly, as shown in FIGs. 19 and 20, B2M knockout Jurkat cells transduced with the mB2M-HLA-E fusion protein exhibited high expression of HLA-E (FIG. 19) and were protected from NK cell mediated cytotoxicity (FIG. 20). The results of the study showed that the mB2M-HLA-E fusion protein construct containing mutated B2M, endogenous B2M signal, and HLA-G leader peptide can be used to generate Allo TRuCs (i.e., T cells with a functional disruption of an endogenous gene encoding a MHC molecule, optionally with functional disruption of an endogenous gene encoding a TCR chain and/or or a subunit thereof, and comprising a TFP as described herein) expressing an NK cell inhibitory signal.

Table 1. Antigen binding domain sequences.

Table 2. TCR sequences

[0614] Anti-MSLN-CD3 epsilon (SEQ ID NO: 195)

MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGGDWSANFMYWYRQAPGKQ

RELVARISGRGVVDYVESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVASYWGQGTLVTVS

SAAAGGGGSGGGGSGGGGSLEDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGG

DEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVI

VDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSG

LNQRRI

Table 3. Exemplary IL-15 or IL-15R sequences

Table 4. Exemplary fusion protein and components

Table 5. Exemplary TFP construct sequences

OTHER EMBODIMENTS

[0615] 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 disclosed 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.

Claims

WHAT IS CLAIMED IS:

1. A modified cell comprising a recombinant nucleic acid comprising a first sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising

(i) a TCR subunit comprising

(1) at least a portion of a TCR extracellular domain, and

(2) a TCR transmembrane domain, and

(ii) an antibody domain comprising an antigen binding domain; and wherein the TCR subunit and the antibody domain are operatively linked, wherein the TFP functionally incorporates into an endogenous TCR complex when expressed in the modified cell, wherein the modified cell comprises a functional disruption of an endogenous major histocompatibility complex (MHC) molecule, wherein the modified cell comprises an enhancing agent or a sequence encoding the enhancing agent that enhances persistence of the modified cell, and wherein the enhancing agent comprises an interleukin- 15 (IL- 15) polypeptide or a fragment thereof.

2. The modified cell of claim 1, wherein the endogenous MHC molecule comprises all endogenous MHC molecules within the modified cell.

3. The modified cell of claim 1 or 2, wherein the endogenous MHC molecule comprises an MHC class I molecule, a MHC class II molecule, or a combination thereof.

4. The modified cell of any one of claims 1-3, wherein the functional disruption of the MHC molecule comprises inactivating a gene encoding the MHC molecule or subunit thereof.

5. The modified cell of claim 4, wherein inactivating the gene encoding the MHC molecule or subunit thereof comprises knocking out or knocking down the gene.

6. The modified cell of claim 4 or 5, wherein the gene encoding the MHC molecule or subunit thereof comprises a gene encoding a beta-2 -microglobulin (B2M) molecule.

7. The modified cell of any one of claims 1-6, wherein the modified cell does not express any MHC molecules on a surface of the modified cell.

8. The modified cell of any one of claims 1-7, wherein the TFP further comprises a TCR intracellular domain.

9. The modified cell of claim 8, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit.

10. The modified cell of claim 9, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR alpha.

11. The modified cell of claim 9, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR beta. The modified cell of claim 9, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma. The modified cell of claim 9, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta. The modified cell of claim 9, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon. The modified cell of claim 9, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta. The modified cell of claim 9, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma. The modified cell of any one of claims 9-16, wherein all three of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. The modified cell of claim 17, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon. The modified cell of claim 17, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta. The modified cell of claim 17, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma. The modified cell of any one of claims 17-20, wherein the recombinant nucleic acid comprises a second sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain or a TCR delta constant domain, or a second sequence encoding a TCR gamma constant domain and a TCR delta constant domain. The modified cell of claim 21, wherein the second sequence further encodes a TCR transmembrane domain, wherein the TCR transmembrane domain is a TCR gamma transmembrane domain or a TCR delta transmembrane domain. The modified cell of claim 21 or 22, wherein the first sequence and the second sequence are contained in a same recombinant nucleic acid molecule. The modified cell of claim 23, wherein the recombinant nucleic acid molecule further comprises a sequence encoding a protease cleavage site. The modified cell of claim 21 or 22, wherein the first sequence and the second sequence are contained in two separate recombinant nucleic acid molecules. The modified cell of claim 17, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR alpha. The modified cell of claim 26, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR alpha. The modified cell of claim 27, wherein the constant domain of TCR alpha is murine. The modified cell of claim 27 or 28, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain further comprise a TCR alpha transmembrane domain and a TCR alpha intracellular domain. The modified cell of any one of claims 27-29, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain do not comprise a variable domain of TCR alpha. The modified cell of claim 17, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR beta. The modified cell of claim 31, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR beta. The modified cell of claim 32, wherein the constant domain of TCR beta is murine. The modified cell of claim 32 or 33, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain further comprise a TCR beta transmembrane domain and a TCR beta intracellular domain. The modified cell of any one of claims 32-34, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain do not comprise a variable domain of TCR beta. The modified cell of claim 17, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma. The modified cell of claim 36, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR gamma. The modified cell of claim 37, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain further comprise a TCR gamma transmembrane domain and a TCR gamma intracellular domain. The modified cell of claim 37 or 38, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain do not comprise a variable domain of TCR gamma. The modified cell of claim 17, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta. The modified cell of claim 40, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR delta. The modified cell of claim 41, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain further comprise a TCR delta transmembrane domain and a TCR delta intracellular domain. The modified cell of claim 41 or 42, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain do not comprise a variable domain of TCR delta. The modified cell of any one of claims 13 and 21-43, wherein the TCR delta or the TCR delta constant domain comprises a sequence of SEQ ID NO: 243.

-164- The modified cell of any one of claims 12 and 21-44, wherein the TCR gamma or the TCR gamma constant domain comprises a sequence of SEQ ID NO: 21. The modified cell of any one of claims 1-43, wherein the modified cell comprises the enhancing agent. The modified cell of any one of claims 1-43, wherein the modified cell comprises the sequence encoding the enhancing agent. The modified cell of claim 47, wherein the recombinant nucleic acid molecule comprises a third sequence that is the sequence encoding the enhancing agent. The modified cell of claim 48, wherein the first sequence and the third sequence are operatively linked by a first linker. The modified cell of claim 49, wherein the first linker comprises a protease cleavage site. The modified cell of claim 50, wherein the protease cleavage site is a 2A cleavage site. The modified cell of any one of claims 46-51, wherein the IL-15 polypeptide is secreted. The modified cell of any one of claims 46-52, wherein the IL-15 polypeptide comprises a sequence of

SEQ ID NO: 385. The modified cell of any one of claims 46-53, wherein the third sequence further encodes an IL-15 receptor (IL-15R) subunit or a fragment thereof. The modified cell of claim 54, wherein the IL-15R subunit is IL-15R alpha (IL-15Ra). The modified cell of claim 54 or 55, wherein IL-15 and IL-15Ra are operatively linked by a second linker. The modified cell of claim 56, wherein the second linker is not a cleavable linker. The modified cell of claim 56 or 57, wherein the second linker comprises a sequence comprising

(G₄S) n, wherein G is glycine, S is serine, and n is an integer from 1 to 10. The modified cell of claim 58, wherein n is an integer from 1 to 4. The modified cell of claim 59, wherein n is 3. The modified cell of any one of claims 56-60, wherein the second linker comprises a sequence of SEQ ID NO: 378 or 405. The modified cell of any one of claims 54-61, wherein the third sequence encodes a fusion protein comprising the IL- 15 polypeptide linked to the IL-15Ra subunit. The modified cell of claim 62, wherein the IL- 15 polypeptide is linked to N-terminus of the IL-15Ra subunit. The modified cell of claim 62 or 63, wherein the fusion protein comprises amino acids 30 - 162 of IL- 15. The modified cell of claim 62 or 63, wherein the fusion protein comprises amino acids 31 - 267 of IL- 15Ra. The modified cell of claim 62 or 63, wherein the fusion protein further comprises a sushi domain.

-165-

67. The modified cell of claim 62 or 63, wherein the fusion protein comprises a sequence of SEQ ID NO: 389.

68. The modified cell of claim 62 or 63, wherein the fusion protein comprises a sequence of SEQ ID NO: 371.

69. The modified cell of any one of claims 62-67, wherein the fusion protein is expressed on cell surface of the modified cell.

70. The modified cell of any one of claims 62-69, wherein the fusion protein is secreted.

71. A modified cell comprising a recombinant nucleic acid comprising a first sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising

(i) a TCR subunit comprising

(1) at least a portion of a TCR extracellular domain, and

(2) a TCR transmembrane domain, and

(ii) an antibody domain comprising an antigen binding domain; and a second sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain or a TCR delta constant domain, or a second sequence encoding a TCR gamma constant domain and a TCR delta constant domain; wherein the TCR subunit and the antibody domain are operatively linked, wherein the TFP functionally incorporates into an endogenous TCR complex when expressed in the modified cell, and wherein the modified cell comprises a functional disruption of an endogenous major histocompatibility complex (MHC) molecule.

72. The modified cell of claim 71, wherein the modified cell comprises an enhancing agent or a sequence encoding the enhancing agent that enhances persistence of the modified cell.

73. The modified cell of claim 72, wherein the modified cell comprises the enhancing agent.

74. The modified cell of claim 72, wherein the modified cell comprises the sequence encoding the enhancing agent.

75. The modified cell of any one of claims 71-74, wherein the recombinant nucleic acid molecule comprises a third sequence that is the sequence encoding the enhancing agent, and wherein the enhancing agent comprises an interleukin- 15 (IL- 15) polypeptide or a fragment thereof.

76. The modified cell of claim 75, wherein the first sequence and the third sequence are operatively linked by a first linker.

77. The modified cell of claim 76, wherein the first linker comprises a protease cleavage site.

78. The modified cell of claim 77, wherein the protease cleavage site is a 2A cleavage site.

79. The modified cell of any one of claims 75-78, wherein the IL-15 polypeptide is secreted.

80. The modified cell of any one of claims 75-78, wherein the third sequence further encodes an IL-15 receptor (IL-15R) subunit or a fragment thereof.

81. The modified cell of claim 80, wherein the IL-15R subunit is IL-15R alpha (IL-15Ra).

-166- The modified cell of claim 80 or 81, wherein IL-15 and IL-15Ra are operatively linked by a second linker. The modified cell of claim 82, wherein the second linker is not a cleavable linker. The modified cell of claim 82 or 83, wherein the second linker comprises a sequence comprising

(G₄S) n, wherein G is glycine, S is serine, and n is an integer from 1 to 10. The modified cell of claim 84, wherein n is an integer from 1 to 4. The modified cell of claim 85, wherein n is 3. The modified cell of any one of claims 75-86, wherein the third sequence encodes a fusion protein comprising the IL- 15 polypeptide linked to the IL-15Ra subunit. The modified cell of claim 87, wherein the IL-15 polypeptide is linked to N-terminus of the IL-15Ra subunit. The modified cell of any one of claims 71-88, wherein the endogenous MHC molecule comprises all endogenous MHC molecules within the modified cell. The modified cell of claim 89, wherein the endogenous MHC molecule comprises an MHC class I molecule, a MHC class II molecule, or a combination thereof. The modified cell of any one of claims 71 -90, wherein the functional disruption of the MHC molecule comprises inactivating a gene encoding the MHC molecule or subunit thereof. The modified cell of claim 91, wherein inactivating the gene encoding the MHC molecule or subunit thereof comprises knocking out or knocking down the gene. The modified cell of claim 91 or 92, wherein the gene encoding the MHC molecule or subunit thereof comprises a gene encoding a beta-2 -microglobulin (B2M) molecule. The modified cell of any one of claims 71-93, wherein the modified cell does not express any MHC molecules on a surface of the modified cell. The modified cell of any one of claims 71-94, wherein the TCR extracellular domain and the TCR transmembrane domain are from a same subunit. The modified cell of claim 95, wherein the same subunit is TCR gamma or TCR delta. The modified cell of any one of claims 71-96, wherein the TCR subunit further comprises a TCR intracellular domain. The modified cell of claim 97, wherein the TCR intracellular domain is from TCR gamma or TCR beta. The modified cell of claim 97 or 98, wherein the TCR extracellular domain, the TCR transmembrane domain and the TCR intracellular domain are from a same subunit. The modified cell of any one of claims 71-99, wherein the second sequence further encodes a second antibody domain comprising a second antigen binding domain. The modified cell of claim 100, wherein the second antigen binding domain and the antigen binding domain are the same. The modified cell of any one of claims 71-101, wherein the first sequence and the second sequence are contained within the same recombinant nucleic acid molecule. The modified cell of any one of claims 71-101, wherein the first sequence and the second sequence are contained within two different recombinant nucleic acid molecules. The modified cell of any one of claims 1-103, wherein the antibody domain is an antibody fragment. The modified cell of claim 104, wherein the antibody fragment is a scFv, a single domain antibody domain, a VH domain or a VL domain. The modified cell of any one of claims 1-105, wherein an antigen binding domain is selected from a group consisting of an anti-mesothelin (MSLN) binding domain, an anti-CD70 binding domain, an anti-Nectin-4 binding domain, and an anti-GPC3 binding domain. The modified cell of claim 106, wherein the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO: 60, a CDR2 of SEQ ID NO: 61, and a CDR3 of SEQ ID NO: 62. The modified cell of claim 106, wherein the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64, and a CDR3 of SEQ ID NO: 65. The modified cell of claim 106, wherein the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO:66, a CDR2 of SEQ ID NO:67, and a CDR3 of SEQ ID NO:68. The modified cell of claim 106, wherein the anti-MSLN binding domain comprises a sequence with at least about 80% sequence identity to a sequence of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71. The modified cell of any one of claims 1-110, wherein the TCR subunit and the antibody domain are operatively linked by a linker. The modified cell of claim 111, wherein the linker comprises a sequence of SEQ ID NO: 387. The modified cell of any one of claims 1-112, wherein the recombinant nucleic acid further comprises a sequence encoding a signal peptide. The modified cell of claim 113, wherein the signal peptide is a GM-CSF signal peptide. The modified cell of any one of claims 1-114, wherein the recombinant nucleic acid molecule further comprises a sequence encoding a protease. The modified cell of claim 115, wherein the protease is a furin. The modified cell of any one of claims 1-116, wherein the recombinant nucleic acid comprises a sequence of SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 400, SEQ ID NO: 402, and/or SEQ ID NO: 404. The modified cell of claim 117, wherein the recombinant nucleic acid molecule comprises a sequence encoding SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 69, SEQ ID NO: 243, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, and/or SEQ ID NO: 21. The modified cell of claim 117 or 118, wherein the recombinant nucleic acid molecule encodes, from N-terminus to C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional anti-MSLN antigen binding domain, operatively linked to a TCR gamma constant domain. The modified cell of any one of claims 1-116, wherein the recombinant nucleic acid comprises a sequence of SEQ ID NO: 407, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 404, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, and/or SEQ ID NO: 411. The modified cell of claim 120, wherein the recombinant nucleic acid molecule comprises a sequence encoding SEQ ID NO: 366, SEQ ID NO: 362, SEQ ID NO: 69, SEQ ID NO: 243, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 385, SEQ ID NO: 405, and/or SEQ ID NO: 403. The modified cell of claim 120 or 121, wherein the recombinant nucleic acid molecule encodes, from N-terminus to C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional anti-MSLN antigen binding domain, operatively linked to a TCR gamma constant domain, operatively linked to a linker, operatively linked to a T2A sequence, operatively linked to a IL- 15 polypeptide, operatively linker to a linker, operatively linked to a hIL-15Ra polypeptide. The modified cell of any one of claims 1-116, wherein the recombinant nucleic acid comprises a sequence of SEQ ID NO: 412, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 413, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 414, and/or SEQ ID NO: 404. The modified cell of claim 123, wherein the recombinant nucleic acid molecule encodes a sequence of SEQ ID NO: 367, SEQ ID NO: 362, SEQ ID NO: 69, SEQ ID NO: 387, SEQ ID NO: 243, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, and/or SEQ ID NO: 21. The modified cell of claim 123 or 124, wherein the recombinant nucleic acid molecule encodes, from N-terminus to C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN antigen binding domain, operatively linked to a first linker, operatively linked to a TCR delta constant domain, operatively linked to fiirin, operatively linked to a second linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional anti-MSLN antigen binding domain, operatively linked to a third linker, operatively linked to a TCR gamma constant domain. The modified cell of any one of claims 1-116, wherein the recombinant nucleic acid comprises a sequence of SEQ ID NO: 415, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 413, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 400, SEQ ID NO: 402, SEQ

-169- ID NO: 414, SEQ ID NO: 404, SEQ ID NO: 390, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, and/or SEQ ID NO: 411. The modified cell of claim 126, wherein the recombinant nucleic acid molecule encodes a sequence of SEQ ID NO: 368, SEQ ID NO: 362, SEQ ID NO: 69, SEQ ID NO: 387, SEQ ID NO: 243, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 385, SEQ ID NO: 405, and/or SEQ ID NO: 403. The modified cell of claim 126 or 127, wherein the recombinant nucleic acid molecule encodes, from N-terminus to C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN antigen binding domain, operatively linked to a first linker, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a second linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional anti-MSLN antigen binding domain, operatively linked to a third linker, operatively linked to a TCR gamma constant domain, operatively linked to a fourth linker, operatively linked to a T2A sequence, operatively linked to a IL- 15 polypeptide, operatively linker to a linker, operatively linked to a hlL- 15Ra polypeptide. The modified cell of any one of claims 1-128, wherein the modified cell comprises a functional disruption of an endogenous TCR chain. The modified cell of claim 129, wherein the endogenous TCR chain 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. The modified cell of claim 129 or 130, wherein the endogenous TCR chain that is functionally disrupted has reduced binding to MHC -peptide complex compared to that of an unmodified control cell. The modified cell of claim 131, wherein the functional disruption is a disruption of a gene encoding the endogenous TCR chain. The modified cell of claim 132, wherein the disruption of a gene encoding the endogenous TCR chain is a removal of a sequence of the gene encoding the endogenous TCR chain from the genome of the modified cell. The modified cell of any one of claims 1-133, wherein the modified cell is a T cell. The modified cell of claim 134, wherein the T cell is a human T cell selected from CD4 cells, CD8 cells, naive T-cells, memory stem T-cells, central memory T- cells, double negative T-cells, effector memory T-cells, effector T-cells, ThO cells, TcO cells, Thl cells, Tel cells, Th2 cells, Tc2 cells,

Th 17 cells, Th22 cells, alpha/beta T cells, gamma/delta T cells, natural killer (NK) cells, natural killer T (NKT) cells, hematopoietic stem cells and pluripotent stem cells. The modified cell of claim 134 or 135, wherein the T cell is a CD8+ or CD4+ T cell. The modified cell of any one of claims 134-136, wherein the T cell is an allogenic T cell.

-170- The modified cell of any one of claims 1-137, further comprising 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. The modified cell of claim 138, wherein 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. A pharmaceutical composition comprising:

(a) the modified cell of any one of claims 1-139; and

(b) a pharmaceutically acceptable carrier. A method of producing the modified cell of any one of claims 1-139, the method comprising

(a) functionally disrupting an endogenous MHC molecule of a cell; and

(b) transducing the cell containing a functional disruption of the endogenous MHC gene with the recombinant nucleic acid of any one of claims 1-139. The method of claim 141, further comprising functionally disrupting an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain; thereby producing a cell containing a functional disruption of an endogenous TCR gene. The method of claim 142, wherein disrupting the endogenous TCR gene comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets the endogenous gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain. The method of any one of claims 141-143, wherein disrupting the endogenous MHC molecule comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets an endogenous gene encoding the endogenous MHC molecule. A method of producing the modified cell of any one of claims 1-139, the method comprising transducing a cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid of any one of claims 1-139. The method of claim 145, wherein the cell containing a functional disruption of an endogenous TCR gene is a cell containing a functional disruption of an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain. The method of claim 145 or 146, wherein the cell further comprises a functional disruption of an endogenous MHC molecule. The method of claim 147, wherein the cell comprises a functional disruption of a gene encoding a B2M molecule. The method of any one of claims 141-146, wherein the cell is a T cell. The method of claim 149, wherein the T cell is a human T cell.

-171- The method of any one of claims 141-149, wherein the cell containing a functional disruption of an endogenous TCR gene has reduced binding to MHC-peptide complex compared to that of an unmodified control cell. The method of any one of claims 143-151, wherein the nuclease protein is a meganuclease, a zinc- finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, or a megaTAL nuclease. The method of any one of claims 141-152, wherein the sequence comprised by the recombinant nucleic acid is inserted into the endogenous TCR subunit gene at the cleavage site, and wherein the insertion of the sequence into the endogenous TCR subunit gene functionally disrupts the endogenous TCR subunit. The method of any one of claims 143-151, wherein the nuclease protein is a meganuclease. The method of claim 154, wherein the meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence, and wherein the second subunit binds to a second recognition half-site of the recognition sequence. The method of claim 155, wherein the meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 140. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a modified cell produced according to the method of any one of claims 141-156; and (b) a pharmaceutically acceptable carrier. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a modified cell produced according to the method of any one of claims 141-156; and (b) a pharmaceutically acceptable carrier. The method of any one of claims 157-159, wherein the modified cell is an allogeneic T cell. The method of any one of claims 157-160, wherein less cytokines are released in the subject compared a subject administered an effective amount of an unmodified control cell. The method of any one of claims 157-161, wherein less cytokines are released in the subject compared a subject administered an effective amount of a modified cell comprising the recombinant nucleic acid of any one of claims 1-139. The method of any one of claims 157-162, wherein the method comprises administering the pharmaceutical composition in combination with an agent that increases the efficacy of the pharmaceutical composition. The method of any one of claims 157-163, wherein the method comprises administering the pharmaceutical composition in combination with an agent that ameliorates one or more side effects associated with the pharmaceutical composition.

-172- The method of any one of claims 157-164, wherein the cancer is a solid cancer, a lymphoma or a leukemia. The method of any one of claims 157-165, wherein the cancer is selected from the group consisting of renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney and stomach cancer. The method of any one of claims 157-166, wherein the cancer is associated with low tumor antigen density. The method of any one of claims 157-167, wherein less cytokines are released in the subject compared to a subject administered an effective amount of an autologous T cell expressing the TFP of claims 1-139. The method of any one of claims 157-168, wherein the method does not induce graft versus host disease. The method of any one of claims 157-169, wherein the subject has a reduced risk of developing graft versus host disease compared to a subject administered an effective amount of an autologous T cell expressing the TFP of claims 1-139. The modified cell of any one of claims 1-139, or the pharmaceutical composition of claim 140, for use as a medicament or in the preparation of a medicament. A modified cell comprising a recombinant nucleic acid comprising a first sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising

(a) a TCR subunit comprising

(i) at least a portion of a TCR extracellular domain, and

(ii) a TCR transmembrane domain, and

(b) an antibody domain comprising an antigen binding domain; wherein the TCR subunit and the antibody domain are operatively linked, wherein the TFP functionally incorporates into an endogenous TCR complex when expressed in the modified cell, wherein the modified cell comprises a functional disruption of an endogenous major histocompatibility complex (MHC) molecule, and wherein the modified cell comprises an agent, or a sequence encoding the agent, that inhibits NK cell activity against the modified cell. The modified cell of claim 172, wherein the agent comprises HLA-E and/or HLA-G. The modified cell of claim 172 or claim 173, wherein the agent is a B2M-HLA-E or B2M-HLA-G fusion protein. The modified cell of any one of claims 172-174, wherein the agent is a fusion protein comprising B2M fused to HLA-E via a Gly-Ser linker. The modified cell of any one of claims 172-175, wherein the B2M is a mutated B2M. The modified cell of claim 176, wherein the mutated B2M comprises a sequence of SEQ ID NO: 420.

-173- The modified cell of claim 177, wherein the agent comprises a HLA-G leader peptide sequence. The modified cell of claim 178, wherein the HLA-G leader peptide is fused to the B2M via a Gly-Ser linker. The modified cell of any one of claims 172-179, wherein the sequence encoding the agent comprises a B2M signal sequence. The modified cell of any one of claims 172-180, wherein the agent comprises a B2M signal sequence, an HLA-G binding protein, a linker, a mutated B2M, a linker, and an HLA-E*01:03. The modified cell of any one of claims 172-181, wherein the agent comprises a sequence according to SEQ ID NO: 423. The modified cell of any one of claims 172-182, wherein the endogenous MHC molecule comprises an MHC class I molecule, a MHC class II molecule, or a combination thereof. The modified cell of any one of claims 172-183, wherein the endogenous MHC molecule comprises all endogenous MHC molecules within the modified cell. The modified cell of any one of claims 172-184, wherein the functional disruption of the MHC molecule comprises inactivating a gene encoding the MHC molecule or subunit thereof. The modified cell of claim 185, wherein inactivating the gene encoding the MHC molecule or subunit thereof comprises knocking out or knocking down the gene. The modified cell of claim 185 or 186, wherein the gene encoding the MHC molecule or subunit thereof comprises a gene encoding a beta-2 -microglobulin (B2M) molecule. The modified cell of any one of claims 172-187, wherein the modified cell does not express any endogenous MHC molecules on a surface of the modified cell. The modified cell of any one of claims 172-188, wherein the modified cell comprises a functional disruption of an endogenous TCR chain selected from TCR alpha and TCR beta. The modified cell of claim 189, wherein the modified cell comprises a functional disruption of the TCR alpha and the TCR beta chains. The modified cell of claim 190, wherein the functional disruption is a disruption of a gene encoding the endogenous TCR chain. The modified cell of claim 191, wherein the disruption of a gene encoding the endogenous TCR chain is a removal of a sequence of the gene encoding the endogenous TCR chain from the genome of the modified cell. The modified cell of any one of claims 172-192, wherein the TFP further comprises a TCR intracellular domain. The modified cell of claim 193, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. The modified cell of claim 193, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma.

-174- The modified cell of claim 193, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta. The modified cell of any one of claims 193-196, wherein all three of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. The modified cell of claim 197, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta. The modified cell of 198, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain do not comprise a variable domain of TCR delta. The modified cell of claim 199, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma. The modified cell of claim 200, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain do not comprise a variable domain of TCR gamma. The modified cell of any one of claims 172-201, wherein the modified cell comprises a second sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain or a TCR delta constant domain, or a second sequence encoding a TCR gamma constant domain and a TCR delta constant domain. The modified cell of claim 202, wherein the second sequence further encodes a TCR transmembrane domain, wherein the TCR transmembrane domain is a TCR gamma transmembrane domain or a TCR delta transmembrane domain. The modified cell of claim 202 or 203, wherein the second sequence further encodes a second antibody domain comprising a second antigen binding domain. The modified cell of claim 204, wherein the second antibody domain and the TCR constant domain are operatively linked. The modified cell of claim 205, wherein the first sequence encodes a TCR delta extracellular domain and TCR delta transmembrane domain, and the second sequence encodes a TCR gamma constant domain and TCR gamma transmembrane domain. The modified cell of any one of claims 204-206, wherein the antibody domain of the first sequence and the antibody domain of the second sequence each comprise the same antigen binding domain. The modified cell of any one of claims 202-207, wherein the first sequence and the second sequence are contained within two different recombinant nucleic acid molecules. The modified cell of any one of claims 202-207, wherein the first sequence and the second sequence are contained in a same recombinant nucleic acid molecule. The modified cell of claim 209, wherein the recombinant nucleic acid molecule further comprises a sequence encoding a protease cleavage site between the first sequence and the second sequence. The modified cell of any one of claims 196, 198, 199, 202, 203, or 206, wherein the TCR delta or the TCR delta constant domain comprises a sequence of SEQ ID NO: 243.

-175- The modified cell of any one of claims 195, 200, 201, 202, 203, or 206, wherein the TCR gamma or the TCR gamma constant domain comprises a sequence of SEQ ID NO: 21 . The modified cell of any one of claims 172-212, wherein recombinant nucleic acid is linked to the sequence encoding the agent by a cleavable linker. The modified cell of claim 213, wherein the cleavable linker comprises a protease cleavage site. The modified cell of claim 214, wherein the protease cleavage site is a 2A cleavage site. The modified cell of any one of claims 172-215, further comprising an enhancing agent, or a sequence encoding the enhancing agent, that enhances persistence of the modified cell, wherein the enhancing agent comprises an IL- 15 or a fragment thereof. The modified cell of claim 216, wherein the enhancing agent comprises an IL-15 receptor (IL-15R) subunit or a fragment thereof. The modified cell of claim 217, wherein the IL-15R subunit is IL-15R alpha (IL-15Ra). The modified cell of claim 218, wherein the IL-15 and the IL-15Ra are operatively linked by a Gly- Ser linker. The modified cell of any one of claims 172-219, wherein the recombinant nucleic acid further comprises a sequence encoding a signal peptide. The modified cell of claim 220, wherein the signal peptide is a GM-CSL signal peptide. The modified cell of any one of claims 172-221, wherein the recombinant nucleic acid molecule further comprises a sequence encoding a protease. The modified cell of claim 222, wherein the protease is a furin. The modified cell of any one of claims 172-223, wherein the sequence encoding the agent is contained within a different recombinant nucleic acid molecule than the recombinant nucleic acid molecule containing the first and second sequences. The modified cell of any one of claims 172-224, wherein the first sequence, the second sequence, and the sequence encoding the agent are contained within the same recombinant nucleic acid molecule. The modified cell of claim 225, wherein the recombinant nucleic acid molecule encodes, from N- terminus to C-terminus, a GM-CSL signal peptide operatively linked to an antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional antigen binding domain, operatively linked to a TCR gamma constant domain, operatively linked to a linker, operatively linked to a T2A sequence, operatively linked to a B2M leader sequence, operatively linked to an HLA-G binding peptide, operatively linked to a linker, operatively linked to a mutB2M, operatively linked to a linker, operatively linked to HLA- E*01:03. The modified cell of claim 225, wherein the recombinant nucleic acid molecule encodes, from N- terminus to C-terminus, a GM-CSF signal peptide operatively linked to an antigen binding domain, operatively linked to a TCR gamma constant domain, operatively linked to furin, operatively linked to

-176- a linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to a linker, operatively linked to a T2A sequence, operatively linked to a B2M leader sequence, operatively linked to an HLA-G binding peptide, operatively linked to a linker, operatively linked to a mutB2M, operatively linked to a linker, operatively linked to HLA-E*01:03. The modified cell of claim 225, wherein the recombinant nucleic acid molecule encodes, from N- terminus to C-terminus, a B2M signal peptide operatively linked to an HLA-G binding peptide, operatively linked to a linker, operatively linked to a mutB2M, operatively linked to a linker, operatively linked to HLA-E*01:03, operatively linked to a T2A sequence, operatively linked to a GM-CSF signal peptide, operatively linked to an antigen binding domain, operatively linked to a TCR delta constant domain, operatively linked to furin, operatively linked to a linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional antigen binding domain, operatively linked to a TCR gamma constant domain. The modified cell of claim 225, wherein the recombinant nucleic acid molecule encodes, from N- terminus to C-terminus, a B2M signal peptide operatively linked to an HLA-G binding peptide, operatively linked to a linker, operatively linked to a mutB2M, operatively linked to a linker, operatively linked to HLA-E*01:03, operatively linked to a T2A sequence, operatively linked to a GM-CSF signal peptide, operatively linked to an antigen binding domain, operatively linked to a TCR gamma constant domain, operatively linked to furin, operatively linked to a linker, operatively linked to a P2A sequence, operatively linked to another GM-CSF signal peptide, operatively linked to an additional antigen binding domain, operatively linked to a TCR delta constant domain. The modified cell of any one of claims 172-229, wherein the antibody domain is an antibody fragment. The modified cell of claim 230, wherein the antibody fragment is a scFv, a single domain antibody domain, a VH domain or a VL domain. The modified cell of any one of claims 172-231, wherein the antigen binding domain is selected from a group consisting of an anti-mesothelin (MSLN) binding domain, an anti-CD70 binding domain, an anti-Nectin-4 binding domain, and an anti-GPC3 binding domain. The modified cell of claim 232, wherein the antigen binding domain is an anti-MSLN binding domain. The modified cell of claim 233, wherein the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO: 60, a CDR2 of SEQ ID NO: 61, and a CDR3 of SEQ ID NO: 62. The modified cell of claim 233, wherein the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64, and a CDR3 of SEQ ID NO: 65. The modified cell of claim 233, wherein the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO:66, a CDR2 of SEQ ID NO:67, and a CDR3 of SEQ ID NO:68.

-177- The modified cell of claim 233, wherein the anti-MSLN binding domain comprises a sequence with at least about 80% sequence identity to a sequence of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71. The modified cell of any one of claims 172-237, wherein the modified cell is a T cell. The modified cell of claim 238, wherein the T cell is a human T cell selected from CD4 cells, CD8 cells, naive T-cells, memory stem T-cells, central memory T- cells, double negative T-cells, effector memory T-cells, effector T-cells, ThO cells, TcO cells, Thl cells, Tel cells, Th2 cells, Tc2 cells,

Th 17 cells, Th22 cells, alpha/beta T cells, gamma/delta T cells, natural killer (NK) cells, natural killer T (NKT) cells, hematopoietic stem cells and pluripotent stem cells. The modified cell of claim 238 or 239, wherein the T cell is a CD8+ or CD4+ T cell. The modified cell of any one of claims 238-240, wherein the T cell is an allogenic T cell. The modified cell of any one of claims 172-241, further comprising 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. The modified cell of claim 242, wherein 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. A pharmaceutical composition comprising:

(a) the modified cell of any one of claims 172-243; and

(b) a pharmaceutically acceptable carrier. A method of producing the modified cell of any one of claims 172-243, the method comprising

(a) functionally disrupting an endogenous MHC molecule of a cell; and

(b) transducing the cell containing a functional disruption of the endogenous MHC gene with the recombinant nucleic acid of any one of claims 1721-243. The method of claim 245, further comprising functionally disrupting an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain; thereby producing a cell containing a functional disruption of an endogenous TCR gene. The method of claim 246, wherein disrupting the endogenous TCR gene comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets the endogenous gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain. The method of claim 246 or 247, wherein disrupting the endogenous MHC molecule comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets an endogenous gene encoding the endogenous MHC molecule. A method of producing the modified cell of any one of claims 172-243, the method comprising transducing a cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid of any one of claims 172-243. The method of claim 249, wherein the cell containing a functional disruption of an endogenous TCR gene is a cell containing a functional disruption of an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain. The method of claim 249 or 250, wherein the cell further comprises a functional disruption of an endogenous MHC molecule. The method of claim 251, wherein the cell comprises a functional disruption of a gene encoding a B2M molecule. The method of any one of claims 249-252, wherein the cell is a T cell. The method of claim 253, wherein the T cell is a human T cell. The method of claim 247 or 248, wherein the nuclease protein is a meganuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, or a megaTAL nuclease. The method of any one of claims 245-255, wherein the sequence comprised by the recombinant nucleic acid is inserted into the endogenous TCR subunit gene at the cleavage site, and wherein the insertion of the sequence into the endogenous TCR subunit gene functionally disrupts the endogenous TCR subunit. The method of claim 255, wherein the nuclease protein is a meganuclease. The method of claim 257, wherein the meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence, and wherein the second subunit binds to a second recognition half-site of the recognition sequence. The method of claim 258, wherein the meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 244. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a modified cell produced according to the method of any one of claims 245-259; and (b) a pharmaceutically acceptable carrier. The method of claim 260 or 261, wherein the modified cell is an allogeneic T cell. The method of any one of claims 260-262, wherein the cancer is a solid cancer, a lymphoma or a leukemia. The method of any one of claims 260-263, wherein the cancer is selected from the group consisting of renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney and stomach cancer. The method of any one of claims 260-264, wherein the cancer is associated with low tumor antigen density. The method of any one of claims 260-265, wherein the method does not induce graft versus host disease. The method of any one of claims 260-266, wherein the method does not elicit an immune response in the subject against the modified cell. The method of any one of claims 260-267, wherein the subject has a reduced risk of rejection of the modified cell compared to a subject administered a modified cell comprising the TFP and that does not comprise the agent. The method of any one of claims 260-268, wherein the subject has a reduced risk of NK cell activity against the modified cell compared to a subject administered a modified cell comprising the TFP and that does not comprise the agent. The modified cell of any one of claims 172-243, or the pharmaceutical composition of claim 244, for use as a medicament or in the preparation of a medicament.