WO2023034220A2 - Compositions and methods for tcr reprogramming using fusion proteins and cxcr6 - Google Patents

Abstract

Provided herein are recombinant nucleic acids comprising a sequence encoding T cell receptor (TCR) fusion proteins (TFPs) and a sequence encoding CXCR6 or a functional fragment thereof, cells comprising the recombinant nucleic acids, methods of using the recombinant nucleic acids and/or the cells, including the methods of treating diseases or conditions, including cancer.

Description

COMPOSITIONS AND METHODS FOR TCR REPROGRAMMING USING FUSION

PROTEINS AND CXCR6

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/238,548, filed August 30, 2021, and U.S. Provisional Patent Application No. 63/318,071, filed March 09, 2022, each of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

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

[0003] Recent developments using chimeric antigen receptor (CAR) modified autologous T cell therapy, which relies on redirecting genetically engineered T cells to a suitable cell-surface molecule on cancer cells, show promising results in harnessing the power of the immune system to treat B cell malignancies (see, e.g., Sadelain et al., Cancer Discovery 3:388-398 (2013)). The clinical results with CD19-specific CAR T cells (called CTL019) have shown complete remissions in patients suffering from chronic lymphocytic leukemia (CLL) as well as in childhood acute lymphoblastic leukemia (ALL) (see, e.g., Kalos et al., Sci Transl Med 3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368: 1509-1518 (2013)). An alternative approach is the use of T cell receptor (TCR) alpha and beta chains selected for a tumor-associated peptide antigen for genetically engineering autologous T cells. These TCR chains will form complete TCR complexes and provide the T cells with a TCR for a second defined specificity. Encouraging results were obtained with engineered autologous T cells expressing NY-ESO-1 -specific TCR alpha and beta chains in patients with synovial carcinoma. [0004] Besides the ability for genetically modified T cells expressing a CAR or a second TCR to recognize and destroy respective target cells in vitro/ex vivo, successful patient therapy with engineered T cells 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.

SUMMARY OF THE INVENTION

[0005] There is a dire 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.

[0006] n one aspect, provided herein are recombinant nucleic acids comprising: a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked, and a second nucleic acid sequence encoding C-X-C chemokine receptor type 6 (CXCR6) or a functional fragment thereof.

[0007] In some embodiments, the TCR subunit further comprises a TCR intracellular domain. [0008] In some embodiments, the first and the second nucleic acid molecules are expressed in the same operon.

[0009] In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a sequence encoding a linker.

[0010] In some embodiments, the linker comprises a protease cleavage site.

[0011] In some embodiments, the protease cleavage site is a 2A cleavage site.

[0012] In some embodiments, the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.

[0013] In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on different nucleic acid molecules.

[0014] In some embodiments, the CXCR6 or functional fragment thereof comprises a sequence with at least 80% sequence identity to any one selected from SEQ ID NOs: 400-402.

[0015] In some embodiments, the CXCR6 or functional fragment thereof comprises a sequence of any one selected from SEQ ID NOs: 400-402.

[0016] In some embodiments, the sequence of the recombinant nucleic acid is codon optimized. [0017] In some embodiments, the CXCR6 or functional fragment thereof is encoded by a nucleic acid with at least 60% sequence identity to SEQ ID NO: 427.

[0018] In some embodiments, the CXCR6 or functional fragment thereof is encoded by the nucleic acid of SEQ ID NO: 427. [0019] In some embodiments, the CXCR6 or functional fragment thereof comprises at least one, two, three, or four extracellular domains.

[0020] In some embodiments, the CXCR6 or functional fragment thereof comprises four extracellular domains.

[0021] In some embodiments, the CXCR6 or functional fragment thereof comprises an N- terminal extracellular region comprising a sequence with at least 80% sequence identity to SEQ ID NO: 403.

[0022] In some embodiments, the CXCR6 or functional fragment thereof comprises an N- terminal extracellular region comprising the sequence of SEQ ID NO: 403.

[0023] In some embodiments, the CXCR6 or functional fragment thereof comprises a CXCL16- binding domain.

[0024] In some embodiments, the CXCR6 or functional fragment thereof is associated with the cell membrane when expressed in a T cell.

[0025] In some embodiments, the CXCR6 or functional fragment thereof comprises a transmembrane region comprising at least one, two, three, four, five, six, or seven transmembrane domains.

[0026] In some embodiments, the transmembrane region comprises the sequence of any one of SEQ ID NOs 409-415, or any combination thereof.

[0027] In some embodiments, the CXCR6 or functional fragment thereof comprises a transmembrane region comprising seven transmembrane domains.

[0028] In some embodiments, the CXCR6 or functional fragment thereof further comprises the sequence of SEQ ID NO 406, the sequence of SEQ ID NO 407, the sequence of SEQ ID NO 408, the sequence of SEQ ID NO 416, the sequence of SEQ ID NO 417, the sequence of SEQ ID NO 418, or any combination thereof.

[0029] In some embodiments, the CXCR6 or functional fragment thereof further comprises the sequence of SEQ ID NO 406, the sequence of SEQ ID NO 407, the sequence of SEQ ID NO 408, or a combination thereof; and the sequence of the sequence of SEQ ID NO 416, the sequence of SEQ ID NO 417, the sequence of SEQ ID NO 418, or any combination thereof. [0030] In some embodiments, the CXCR6 or functional fragment thereof comprises a transmembrane region comprising a sequence with at least 80% sequence identity to SEQ ID NO: 428.

[0031] In some embodiments, the CXCR6 or functional fragment thereof comprises a transmembrane region comprising the sequence of SEQ ID NO: 428. [0032] In some embodiments, the CXCR6 or functional fragment thereof comprises at least one, two, three, or four cytoplasmic domains.

[0033] In some embodiments, the CXCR6 or functional fragment thereof comprises four cytoplasmic domains.

[0034] In some embodiments, the CXCR6 or functional fragment thereof comprises a C- terminal cytoplasmic domain comprising a sequence with at least 80% sequence identity to SEQ ID NO: 419.

[0035] In some embodiments, the CXCR6 or functional fragment thereof comprises a cytoplasmic domain comprising the sequence of SEQ ID NO: 419.

[0036] In some embodiments, migration of a cell expressing the CXCR6 or functional fragment thereof increases in response to CXCL16.

[0037] In some embodiments, (i) a migration rate of a cell expressing the CXCR6 or functional fragment thereof increases in response to CXCL16 , (ii) the number of cells expressing the CXCR6 or functional fragment thereof that migrate to a tumor site increases in response to CXCL16, or (iii) a combination thereof.

[0038] In some embodiments, the recombinant nucleic acid as provided herein comprises a sequence encoding an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 420, SEQ ID NO: 424, SEQ ID NO: 426, or SEQ ID NO: 435.

[0039] In some embodiments, the recombinant nucleic acid as provided herein comprises a sequence encoding the sequence of SEQ ID NO: 420, SEQ ID NO: 424, SEQ ID NO: 426, or SEQ ID NO: 435.

[0040] In some embodiments, the TFP functionally interacts with an endogenous TCR complex when expressed in a T cell.

[0041] In some embodiments, the TCR intracellular domain comprises a stimulatory domain from an intracellular signaling domain of CD3 gamma, CD3 delta, or CD3 epsilon.

[0042] In some embodiments, the TCR intracellular domain comprises an intracellular domain from TCR alpha, TCR beta, TCR gamma, or TCR delta.

[0043] In some embodiments, the antigen binding domain is connected to the TCR extracellular domain by a linker sequence.

[0044] In some embodiments, the linker sequence is 120 amino acids in length or less.

[0045] In some embodiments, the linker sequence comprises (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 10.

[0046] In some embodiments, n is an integer from 1 to 4. [0047] 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. [0048] In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR alpha.

[0049] In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR beta.

[0050] In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma.

[0051] In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta.

[0052] In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon.

[0053] In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta.

[0054] In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma.

[0055] 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.

[0056] In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon.

[0057] In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta.

[0058] In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma.

[0059] In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR alpha.

[0060] In some embodiments, the constant domain of TCR alpha is murine.

[0061] In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR beta.

[0062] In some embodiments, the constant domain of TCR beta is murine.

[0063] In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR gamma.

[0064] In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR delta. [0065] In some embodiments, the antigen binding domain is a camelid antibody or binding fragment thereof.

[0066] In some embodiments, the antigen binding domain is a murine antibody or binding fragment thereof.

[0067] In some embodiments, the antigen binding domain is a human or humanized antibody or binding fragment thereof.

[0068] In some embodiments, the antigen binding domain is a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain.

[0069] In some embodiments, the antigen binding domain is a single domain antibody (sdAb). [0070] In some embodiments, the sdAb is a VH or VHH.

[0071] In some embodiments, the antigen binding domain is selected from the group consisting of an anti-CD19 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, and an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, anti-MUC16 binding domain, an anti-Nectin-4 binding domain, an anti-GPC3 binding domain, and an anti-TROP-2 binding domain.

[0072] In some embodiments, a T cell expressing the TFP inhibits tumor growth.

[0073] In some embodiments, the recombinant nucleic acid as provided herein further comprises a leader sequence.

[0074] In some embodiments, the recombinant nucleic acid comprises a sequence encoding (i) a TFP comprising a GM-CSFRa signal peptide, an anti-MSLN scFv or VHH antibody or a fragment thereof, a linker, a CD3 epsilon intracellular signaling domain, and (ii) the CXCR6 or fragment thereof.

[0075] In some embodiments, the recombinant nucleic acid comprises (i) a sequence encoding a TFP comprising, from the N-terminus to the C-terminus, the GM-CSFRa signal peptide operatively linked to the anti-MSLN scFv or VHH antibody or fragment thereof operatively linked to the linker operatively linked to the CD3 epsilon intracellular signaling domain and (ii) a sequence encoding CXCR6 or fragment thereof, or (i) the sequence encoding CXCR6 or fragment thereof and (ii) the sequence encoding the TFP comprising the GM-CSFRa signal peptide operatively linked to the anti-MSLN scFv or VHH antibody or fragment thereof operatively linked to the linker operatively linked to the CD3 epsilon intracellular signaling domain, wherein CXCR6 and the TFP are expressed in the same operon and are separated by a cleavable linker.

[0076] In some embodiments, the linker is a A3(G4S)3LE linker. [0077] In some embodiments, from the N-terminus to the C-terminus, the CD3 epsilon intracellular signaling domain is operatively linked to the CXCR6 or fragment thereof via a cleavable linker or the CXCR6 or fragment thereof is operatively linked to the GM-CSFRa signal peptide via the cleavable linker.

[0078] In some embodiments, the cleavable linker is a 2A cleavage site or a furin cleavage site. [0079] In some embodiments, the 2A cleavage site is a P2A cleavage site or a T2A cleavage site.

[0080] In some embodiments, the recombinant nucleic acid encodes a sequence comprising the sequences of SEQ ID NOs: 421, 422, 423, and 400.

[0081] In some embodiments, the recombinant nucleic acid encodes a sequence comprising, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 421 operatively linked to the sequence of SEQ ID NO: 422 operatively linked to the sequence of SEQ ID NO: 423 operatively linked to the sequence of SEQ ID NO: 400, or the sequence of SEQ ID NO: 400 operatively linked to the sequence of SEQ ID NO: 421 operatively linked to the sequence of SEQ ID NO: 422 operatively linked to the linker operatively linked to the sequence of SEQ ID NO: 423.

[0082] In some embodiments, the recombinant nucleic acid comprises a sequence encoding, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 422 is operatively linked to the sequence of SEQ ID NO: 423 via the sequence of SEQ ID NO: 387.

[0083] In some embodiments, the recombinant nucleic acid comprises a sequence encoding, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 423 is operatively linked to the sequence of SEQ ID NO: 400 via the sequence of SEQ ID NO: 23 or the sequence of SEQ ID NO 425 operatively linked to the sequence of SEQ ID NO: 23, or the sequence of SEQ ID NO: 400 is operatively linked to the sequence of SEQ ID NO: 421 via the sequence of SEQ ID NO: 425 linked to the sequence of SEQ ID NO: 23.

[0084] In some embodiments, the recombinant nucleic acid as provided herein further comprises a third nucleic acid sequence.

[0085] In some embodiments, the third nucleic acid sequence encodes a switch polypeptide comprising a transforming growth factor beta receptor II (TGFBr2) extracellular domain or a functional fragment thereof.

[0086] In some embodiments, the TGFBr2 extracellular domain or functional fragment thereof comprises a sequence with at least 80% sequence identity to SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437. [0087] In some embodiments, the TGFBr2 extracellular domain or functional fragment thereof comprises the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437.

[0088] In some embodiments, the switch polypeptide further comprises a switch intracellular domain.

[0089] In some embodiments, the TGFBr2 extracellular domain or functional fragment thereof is operably linked to the switch intracellular domain.

[0090] In some embodiments, the switch intracellular domain comprises an intracellular domain of a costimulatory polypeptide.

[0091] In some embodiments, the costimulatory polypeptide is selected from the group consisting of CD28, 4-1BB, IL-15Ra, 0X40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, and FcyRIII.

[0092] In some embodiments, the costimulatory polypeptide is CD28.

[0093] In some embodiments, the costimulatory polypeptide is 4- IBB.

[0094] In some embodiments, the costimulatory polypeptide is IL-15Ra.

[0095] In some embodiments, the switch intracellular domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:273 or SEQ ID NO:277.

[0096] In some embodiments, the switch intracellular domain comprises the sequence of SEQ ID NO:273 or SEQ ID NO:277.

[0097] In some embodiments, the switch polypeptide further comprises a switch transmembrane domain.

[0098] In some embodiments, the TGFBr2 extracellular domain or functional fragment thereof is operably linked to the switch intracellular domain via the switch transmembrane domain.

[0099] In some embodiments, the switch transmembrane domain is a TGFBr2 transmembrane domain.

[0100] In some embodiments, the switch transmembrane domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:272.

[0101] In some embodiments, the switch transmembrane domain comprises the sequence of SEQ ID NO:272.

[0102] In some embodiments, the switch transmembrane domain is a transmembrane domain of the costimulatory polypeptide.

[0103] In some embodiments, the switch transmembrane domain is a transmembrane domain of CD28. [0104] In some embodiments, the switch transmembrane domain is a transmembrane domain of 4-1BB.

[0105] In some embodiments, the switch transmembrane domain is a transmembrane domain of IL-15Ra.

[0106] In some embodiments, the switch transmembrane domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:275 or SEQ ID NO:279.

[0107] In some embodiments, the switch transmembrane domain comprises the sequence of SEQ ID NO:275 or SEQ ID NO:279.

[0108] In some embodiments, the switch polypeptide further comprises an additional intracellular domain.

[0109] In some embodiments, the additional intracellular domain is operably linked to the C- terminus of the switch intracellular domain.

[0110] In some embodiments, the additional intracellular domain comprises an intracellular domain of IL-15Ra or signaling domain thereof.

[OHl] In some embodiments, the additional intracellular domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:372 or SEQ ID NO:383.

[0112] In some embodiments, the additional intracellular domain comprises the sequence of SEQ ID NO:372 or SEQ ID NO:383.

[0113] In some embodiments, the switch polypeptide comprises a TGFBr2 transmembrane domain and an intracellular signaling domain of 4-1BB.

[0114] In some embodiments, the switch polypeptide comprises a 4-1BB transmembrane domain and an intracellular signaling domain of 4-1BB.

[0115] In some embodiments, the switch polypeptide comprises a TGFBr2 transmembrane domain and an intracellular signaling domain of CD28.

[0116] In some embodiments, the switch polypeptide comprises a CD28 transmembrane domain and an intracellular signaling domain of CD28.

[0117] In some embodiments, the switch polypeptide comprises a sequence with at least 80% sequence identity to any one selected from SEQ ID NOs: 283, 284, 285, and 286.

[0118] In some embodiments, the switch polypeptide comprises the sequence of SEQ ID NOs: 283, 284, 285, or 286.

[0119] In some embodiments, the third nucleic acid sequence encodes a dominant negative TGFBR2 receptor or a fragment thereof. [0120] In some embodiments, the dominant negative TGFBR2 receptor or a fragment thereof comprises a sequence with at least 80% sequence identity to SEQ ID NO: 433 or SEQ ID NO: 434.

[0121] In some embodiments, the dominant negative TGFBR2 receptor or a fragment thereof comprises the sequence of SEQ ID NO: 433 or SEQ ID NO: 434.

[0122] In some embodiments, the third nucleic acid sequence encodes an interleukin- 15 (IL- 15) polypeptide or a fragment thereof.

[0123] In some embodiments, expression of the IL- 15 polypeptide or fragment thereof increases persistence of a cell expressing the IL-15 polypeptide or fragment thereof.

[0124] In some embodiments, the IL- 15 polypeptide or fragment thereof is secreted when expressed in a cell.

[0125] In some embodiments, the IL- 15 polypeptide or fragment thereof comprises a sequence with at least 80% sequence identity to SEQ ID NO: 1242 or SEQ ID NO: 1245.

[0126] In some embodiments, the IL- 15 polypeptide or fragment thereof comprises the sequence of SEQ ID NO: 1242 or SEQ ID NO: 1245.

[0127] In some embodiments, the third nucleic acid sequence further encodes an IL-15 receptor (IL-15R) subunit or a fragment thereof.

[0128] In some embodiments, the IL-15R subunit is IL-15R alpha (IL-15Ra).

[0129] In some embodiments, the IL-15 polypeptide or fragment thereof and the IL-15Ra are operatively linked by a second linker.

[0130] In some embodiments, the second linker is not a cleavable linker.

[0131] 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.

[0132] In some embodiments, n is an integer from 1 to 4.

[0133] In some embodiments, n is 3.

[0134] In some embodiments, the second linker comprises the sequence of SEQ ID NO: 1243.

[0135] In some embodiments, the third nucleic acid sequence encodes a fusion protein comprising the IL-15 polypeptide or fragment thereof linked to the IL-15Ra subunit.

[0136] In some embodiments, the IL- 15 polypeptide or fragment thereof is linked to N-terminus of the IL-15Ra subunit.

[0137] In some embodiments, the fusion protein comprises amino acids 30 - 162 of IL-15. [0138] In some embodiments, the fusion protein comprises amino acids 31 - 267 of IL-15Ra. [0139] In some embodiments, the fusion protein further comprises a sushi domain. [0140] In some embodiments, the fusion protein comprises a sequence with at least 80% sequence identity to SEQ ID NO: 1253.

[0141] In some embodiments, the fusion protein comprises the sequence of SEQ ID NO: 1253. [0142] In some embodiments, the fusion protein is expressed on cell surface when expressed in a cell.

[0143] In some embodiments, the fusion protein is secreted when expressed in a cell.

[0144] In some embodiments, the third nucleic acid sequence encodes a PD-1 polypeptide or a fragment thereof.

[0145] In some embodiments, the PD-1 polypeptide or fragment thereof is operably linked via its C-terminus to the N-terminus of an intracellular domain of a costimulatory polypeptide. [0146] In some embodiments, the PD-1 polypeptide or fragment thereof is linked to the intracellular domain of the costimulatory polypeptide via a transmembrane domain of PD-1.

[0147] In some embodiments, the costimulatory polypeptide is chosen from a group comprising 0X40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, and FcyRIII.

[0148] In some embodiments, the intracellular domain of the costimulatory polypeptide comprises at least a portion of CD28.

[0149] In some embodiments, an extracellular domain and a transmembrane domain of PD-1 are linked to an intracellular domain of CD28.

[0150] In some embodiments, the third nucleic acid sequence encodes a fusion protein comprising the extracellular domain and the transmembrane domain of PD-1 are linked to the intracellular domain of CD28.

[0151] In some embodiments, the fusion protein comprises a sequence with at least 80% sequence identity to SEQ ID NO: 1239 or SEQ ID NO: 1244.

[0152] In some embodiments, the fusion protein comprises the sequence of SEQ ID NO: 1239 or SEQ ID NO: 1244.

[0153] In some embodiments, the third nucleic acid sequence encodes a fusion protein comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15Ra.

[0154] In some embodiments, the fusion protein comprises a sequence with at least 80% sequence identity to SEQ ID NO: 1254 or SEQ ID NO: 1262.

[0155] In some embodiments, the fusion protein comprises the sequence of SEQ ID NO: 1254 or SEQ ID NO: 1262. [0156] In some embodiments, the third nucleic acid sequence is operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof.

[0157] In some embodiments, the third nucleic acid sequence is operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof by a sequence encoding a third linker.

[0158] In some embodiments, the third linker comprises a protease cleavage site.

[0159] In some embodiments, the protease cleavage site is a 2A cleavage site.

[0160] In some embodiments, the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.

[0161] In some embodiments, the third nucleic acid sequence and the first nucleic acid sequence, the third nucleic acid sequence and the second nucleic acid sequence, or the third nucleic acid sequence, the first nucleic acid sequence, and the second nucleic acid sequence are present on different nucleic acid molecules.

[0162] In some embodiments, the recombinant nucleic acid as provided herein further comprises a fourth nucleic acid sequence.

[0163] In some embodiments, the fourth nucleic acid sequence encodes a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof, a dominant negative TGFBR2 receptor or a fragment thereof, an IL- 15 polypeptide or a fragment thereof, an IL- 15 polypeptide or a fragment thereof operatively linked to an IL-15R subunit or a fragment thereof, a PD-1 polypeptide or a fragment thereof, or a fusion protein comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15Ra.

[0164] In some embodiments, the fourth nucleic acid sequence encodes a polypeptide having a sequence with at least 80% sequence identity to any one selected from SEQ ID NOs: 283, 284, 285, 286, 433, 434, 1242, 1245, 1253, 1239, 1244, 1254, and 1262.

[0165] In some embodiments, the fourth nucleic acid sequence encodes a polypeptide having the sequence of SEQ ID NOs: 283, 284, 285, 286, 433, 434, 1242, 1245, 1253, 1239, 1244, 1254, or 1262.

[0166] In some embodiments, the fourth nucleic acid sequence encodes a polypeptide different from a polypeptide encoded by the third nucleic acid sequence.

[0167] In some embodiments, the fourth nucleic acid sequence is operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, or a combination thereof. [0168] In some embodiments, the third nucleic acid sequence is operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence by a sequence encoding a fourth linker.

[0169] In some embodiments, the fourth linker comprises a protease cleavage site.

[0170] In some embodiments, the protease cleavage site is a 2A cleavage site.

[0171] In some embodiments, the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.

[0172] In some embodiments, the fourth nucleic acid sequence and the first nucleic acid sequence, the fourth nucleic acid sequence and the second nucleic acid sequence, the fourth nucleic acid sequence and the third nucleic acid sequence, or the fourth nucleic acid sequence, the first nucleic acid sequence, and the second nucleic acid sequence, and the third nucleic acid sequence are present on different nucleic acid molecules.

[0173] In some embodiments, the recombinant nucleic acid is selected from the group consisting of a DNA and an RNA.

[0174] In some embodiments, the recombinant nucleic acid is an mRNA.

[0175] In some embodiments, the recombinant nucleic acid is a circRNA.

[0176] In some embodiments, the recombinant nucleic acid comprises a nucleotide analog.

[0177] In some embodiments, the nucleotide analog is selected from the group consisting of 2’- O-methyl, 2’-O-methoxyethyl (2’-0-M0E), 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 ’ -phosphorami di te.

[0178] In some embodiments, the recombinant nucleic acid as provided herein further comprises a promoter.

[0179] In some embodiments, the recombinant nucleic acid is an in vitro transcribed nucleic acid.

[0180] In some embodiments, the recombinant nucleic acid as provided herein further comprises a sequence encoding a poly (A) tail.

[0181] In some embodiments, the recombinant nucleic acid as provided herein further comprises a 3’UTR sequence. [0182] In another aspect, provided herein are polypeptides encoded by the recombinant nucleic acid as provided herein.

[0183] In another aspect, provided herein are vectors comprising a recombinant nucleic acid as provided herein.

[0184] In some embodiments, the vector is a lentiviral vector.

[0185] In another aspect, provided herein are cells comprising the recombinant nucleic acid as provided herein, the polypeptide as provided herein, or the vector as provided herein.

[0186] In some embodiments, the cell is a T cell.

[0187] In some embodiments, the T cell is a human T cell.

[0188] In some embodiments, the T cell is a CD8+ or CD4+ T cell.

[0189] In some embodiments, the T cell is a human aP T cell.

[0190] In some embodiments, the T cell is a human y6 T cell.

[0191] In some embodiments, the cell is a human NKT cell.

[0192] In some embodiments, the cell is an allogeneic cell or an autologous cell.

[0193] In some embodiments, the cell has increased anti-tumor efficacy compared to the antitumor efficacy of a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence.

[0194] In some embodiments, the cell has enhanced migration compared to migration of a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence.

[0195] In some embodiments, in response to CXCL16 (i) the cell has a higher migration rate compared to a migration rate of a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence, (ii) more of the cells migrate to a tumor in response to CXCL16 compared to the number of cells comprising the first nucleic acid sequence and not comprising the second nucleic acid sequence that migrate to a tumor, or (iii) a combination thereof.

[0196] In some embodiments, the cell has enhanced tumor lysis activity compared to a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence.

[0197] In some embodiments, the cell has increased cytokine production compared to a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence.

[0198] In some embodiments, the cell comprises a population of cells. [0199] In some embodiments, the population of cells comprises at least lxl0^A5 cells or at least lxlO^A6 cells.

[0200] In another aspect, provided herein are pharmaceutical compositions comprising the cell as provided herein and a pharmaceutically acceptable carrier.

[0201] In another aspect, provided herein are methods of increasing an activity of a cell expressing a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP), the method comprising expressing CXCR6 or a functional fragment thereof in the cell: wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked.

[0202] In some embodiments, the cell is the cell as provided herein.

[0203] In some embodiments, the cell has enhanced migration compared to migration of a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof.

[0204] In some embodiments, (i) the migration rate of the cell in response to CXCL16 is faster compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof, (ii) more number of the cell migrates in response to CXCL16 compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof, or (iii) a combination thereof.

[0205] In some embodiments, the cell has enhanced tumor lysis activity compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof.

[0206] In some embodiments, the cell has increased cytokine production compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof.

[0207] In another aspect, provided herein are methods of enhancing migration of a cell expressing a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP), the method comprising expressing CXCR6 or a functional fragment thereof in the cell: wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked.

[0208] In some embodiments, the cell is the cell as provided herein. [0209] In some embodiments, (i) the migration rate of the cell in response to CXCL16 is faster compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof, (ii) more number of the cell migrates in response to CXCL16 compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof, or (iii) a combination thereof.

[0210] In another aspect, provided herein are methods of enhancing tumor lysis activity of a cell expressing a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP), the method comprising expressing CXCR6 or a functional fragment thereof in the cell: wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked.

[0211] In some embodiments, the cell is the cell as provided herein.

[0212] In another aspect, provided herein are methods of increasing cytokine production by a cell expressing a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP), the method comprising expressing CXCR6 or a functional fragment thereof in the cell: wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked.

[0213] In some embodiments, the cell is the cell as provided herein.

[0214] In another aspect, provided herein are methods 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 composition as described herein.

[0215] 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.

[0216] In some embodiments, the cancer is a hematologic cancer selected from the group consisting of B-cell acute lymphoid leukemia (B-ALL), T cell acute lymphoid leukemia (T- ALL), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell-follicular lymphoma, large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin’s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and preleukemia.

[0217] In some embodiments, the cancer is mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, thyroid cancer, bladder cancer, ureter cancer, kidney cancer, endometrial cancer, esophageal cancer, gastric cancer, thymic carcinoma or cholangiocarcinoma.

[0218] In some embodiments, the subject is a human.

INCORPORATION BY REFERENCE

[0219] 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

[0220] 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 of which:

[0221] FIG 1 is a graph showing the results of RNA-seq analysis on tumor samples from 33 different tumor types showing CXCL16 expression levels.

[0222] FIG. 2 is a series of plots showing cell surface expression of MH1 TFPs and cell surface and intracellular expression of CXCR6, as determined by flow cytometry.

[0223] FIG. 3 is a series of plots showing the memory phenotype (top) and CD4:CD8 T cell distribution (bottom) and of cells transduced with the TFPs as determined by flow cytometry. All plots are gated on CD3+ cells.

[0224] FIG. 4 is a schematic illustration of the transwell migration assay described in Example 3.

[0225] FIG. 5 is a series of graphs showing the proportion of cells expressing MH1 TFPs with or without CXCR6 that have migrated to the lower well of the transwell plate containing the concentrations of CXCL16 shown in the transwell migration assay described in Example 3 after 4 or 15 hours of incubation. Here, when CXCR6 is present, it is positioned upstream of the MH1 TFP on the lentiviral vector.

[0226] FIG 6 is a series of graphs showing the proportion of cells expressing MH1 TFPs with or without CXCR6 that have migrated to the lower well of the transwell plate containing the concentrations of CXCL16 shown in the transwell migration assay described in Example 3 after 4 hours of incubation.

[0227] FIG. 7 is a series of graphs the proportion of cells expressing MH1 TFPs with or without CXCR6 that have migrated to the lower well of the transwell plate containing supernatant of the cancer cell lines shown in the transwell migration assay described in Example 3 after 4 hours of incubation. Here, when CXCR6 is present, it is positioned upstream of the MH1 TFP on the lentiviral vector. Also shown in the level of soluble CXCL16 expression produced by each of the cancer cell lines.

[0228] FIG. 8 is a series of graphs showing cytotoxicity of T cells expressing the TFP constructs shown (with or without CXCR6) when contacted with MSTO-msln, OVCAR3, Suit- 2, or Panc-1 target cells, as described in Example 4.

[0229] FIGs. 9A-9D is a series of graphs showing cytokine expression (fFNy, GM-CSF, IL-2, and TNF-a) by T cells expressing the TFP constructs shown (with or without CXCR6) when contacted with MSTO-msln, OVCAR3, Suit-2, or Panc-1 target cells, as described in Example 5. FIG. 9A shows GM-CSF expression. FIG. 9B shows ZFNy expression. FIG. 9C shows IL-2 expression. FIG. 9D shows TNF-a expression.

[0230] FIG. 10 is a series of flow cytometry plots showing cell surface expression of CXCR6 on VHH+ (top row) and VHH- (bottom row) cells in each of the indicated groups.

[0231] FIG. 11 is a series of graphs showing the total CD3+ count; total VHH+ count; and %VHH+ cells that migrated to the lower well of the transwell plate containing the indicated concentration of CXCL16.

[0232] FIG. 12 is a series of graphs showing the total CD3+ count; total VHH+ count; and %VHH+ of cells that migrated to the lower well of the transwell plate containing 25 or 50 ng/mL CXCL16, in the presence or absence of CXCL16 blocking antibody.

[0233] FIG. 13 is a series of graphs showing the total CD3+ count; VHH MFI; and % VHH+ of cells that migrated to the lower well of the transwell plate containing MSTO-CXCL16 cells, at the indicated tumor cell:T cell ratio.

[0234] FIGs. 14A-14D is a series of graphs showing the % tumor lysis in the transwell plate migration and killing assay, at the indicated E:T, when MSTO-msln (FIG. 14A), Suit-2 (FIG. 14B), MSTO-CXCL16 (FIG. 14C), and MSTO-msln-CXCL16 (FIG. 14D) cells were used as the target cells.

[0235] FIGs. 15A-15D is a series of graphs showing the % of mouse CD45+ cells in the tumor (FIG. 15A) and of human CD45+ cells in the tumor (FIG. 15B), and the % of human CD45+ cells found in the spleen (FIG. 15C) and liver (FIG. 15D), as determined on Day 4 and Day 7 after administration of the CXCR6 TFP or control cells.

[0236] FIG. 16 is a series of graphs showing the total VHH+ cell count/mg tissue in the tumor, spleen, and liver of mice at Day 4 and Day 7 after administration of CXCR6 TFP or control cells. All groups except the NT group were gated on VHH+ (NT gated on total hCD45+).

[0237] FIG. 17 is a set of graphs showing the total number of VHH+ cells per tumor at Day 4 and Day 7 after administration of CXCR6 TFP or control cells. All groups except the NT group were gated on VHH+ (NT gated on total hCD45+).

[0238] FIG. 18 is a series of graphs showing the %VHH+ cells in the tumor, spleen, and liver at Day 4 and Day 7.

[0239] FIG. 19 is a series of graphs showing the % of VHH+ cells that are CXCR6+ in each group at Days 0, 4, and 7. All groups except the NT group were gated on VHH+ (NT gated on total hCD45+).

[0240] FIG. 20 is a series of graphs showing the % of VHH+ cells that are Ki67+ in each group at Days 0, 4, and 7. All groups except the NT group were gated on VHH+ (NT gated on total hCD45+).

[0241] FIG. 21 is a series of graphs showing the CD4:CD8 ratio of VHH+ cells in the tumor, spleen, and liver at Days 0, 4, and 7.

[0242] FIG. 22A and FIG. 22B provide a series of graphs showing the memory phenotype of VHH+ CD4+ (FIG. 22A) and VHH+ CD8+ (FIG. 22B) cells in the tumors of mice at Days 0, 4, and 7.

DETAILED DESCRIPTION OF THE INVENTION

[0243] The present disclosure provides T cells expressing a T cell receptor (TCR) fusion protein (TFP) and CXCR6 or a functional fragment thereof. The TFP and CXCR6 can be expressed from the same or different nucleic acid molecules. The disclosure also provides a recombinant nucleic acid comprising a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) and a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof. The TFP can comprise (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain. The TCR subunit and the antigen binding domain can be operatively linked. The first nucleic acid sequence and the second nucleic acid sequence can be present on the same or different nucleic acid molecules.

[0244] The present disclosure further provides a vector comprising the recombinant nucleic acid, a cell comprising the recombinant nucleic acid or the vector described herein, or a pharmaceutical composition comprising the cell (e.g., modified cell).

[0245] The present disclosure also provides a method of increasing migration of a cell expressing a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP). The method can comprise expressing CXCR6 or a functional fragment thereof in the cell. The present disclosure also provides a method of treating a disease such as cancer using the cell described herein. The present disclosure further provides a method of enhancing tumor lysis activity of a cell expressing a recombinant nucleic acid comprising a sequence encoding a TFP, or a method of increasing cytokine production by a cell expressing a recombinant nucleic acid comprising a sequence encoding a TFP.

[0246] As is described above, CXCR6 binds the ligand CXCL16. CXCL16 has previously been shown to be highly expressed by pancreatic cancer cells. Herein, we have shown that CXCL16 is highly expressed on a wide variety of different tumor types in addition to pancreatic tumors. CXCL16 can be present as a surface bound molecule or secreted into the surrounding tissue. The secreted form of CXCL16 acts as a chemoattractant and induces proliferation and migration of cancer cells. It has surprisingly been shown herein that expression of CXCR6 in TFP expressing T cells allows migration of the T cells towards a CXCL16 chemokine gradient and towards the supernatant of a variety of types of tumor cells secreting CXCL16. Moreover, it is noted that CXCL16 is the only ligand for CXCR6, which is unusual in the chemokine/receptor family and ensures that CXCR6 expressing TFP T cells will only be targeted to CXCL16 expressing cells, and not to cells expressing other cytokines. Without wishing to be bound by theory, the discoveries presented herein show that that expression of CXCR6 in TFP expressing T cells facilitates migration of the T cells into the tumor microenvironment where CXCL16 is present, thereby increasing the efficacy of TFP-expressing T cells in treating cancer and solid tumors, in particular.

Definitions

[0247] 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. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.

[0248] 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.

[0249] As used herein, the term “comprise” or variations thereof such as “comprises” or “comprising” are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein, the term “comprising,” is inclusive and does not exclude additional, unrecited integers or method/process steps.

[0250] In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of’. The phrase “consisting essentially of’ is used herein to require the specified integer(s) or steps as well as those which do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) alone.

[0251] 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.

[0252] 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. The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human). [0253] 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.

[0254] 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.

[0255] As used herein, the terms “modulate” and “modulation” refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.

[0256] 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)). [0257] As used herein, “chemokines” or “chemotactic cytokines” refer to a family of small cytokines or signaling proteins secreted by cells, which induce directed chemotaxis in nearby responsive cells. In some embodiments, the chemokine is approximately 8-10 kilodaltons in mass. In some embodiments, the chemokine has four cysteine residues in conserved locations that are key to forming their 3-dimensional shape. In some embodiments, the chemokine is classified into one of the four main subfamilies: CXC, CC, CX3C and XC. In some embodiments, the chemokine acts as a chemoattractant to guide the migration of cells. In some embodiments, the chemokines exert their biological effects by interacting with G protein-linked transmembrane receptors called chemokine receptors, which are selectively found on the surfaces of their target cells. [0258] In some embodiments, the chemokine is considered homeostatic and involved in controlling the migration of cells during normal processes of tissue maintenance or development. In some embodiments, the homeostatic chemokine controls cells of the immune system during processes of immune surveillance, for example, directing lymphocytes to the lymph nodes so they can screen for invasion of pathogens by interacting with antigen-presenting cells residing in these tissues. In some embodiments, the homeostatic chemokine is produced and secreted without any need to stimulate their source cells. In some embodiments, the homeostatic chemokine is constitutively produced in certain tissues. In some embodiments, the homeostatic chemokine is responsible for basal leukocyte migration. In some embodiments, the chemokine has roles in development. In some embodiments, the chemokine promotes angiogenesis, i.e., the growth of new blood vessels. In some embodiments, the chemokine guides cells to tissues that provide specific signals critical for cellular maturation. Exemplary homeostatic chemokines include, but are not limited to, CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12 and CXCL13.

[0259] In some embodiments, the chemokine is pro-inflammatory and induced during an immune response to recruit cells of the immune system to a site of infection. In some embodiments, the inflammatory chemokine is formed under pathological conditions, for example, on pro-inflammatory stimuli, such as TNF-alpha, LPS, and viruses. In some embodiments, release of the inflammatory chemokine is stimulated by pro-inflammatory cytokines, for example, interleukin 1. In some embodiments, the inflammatory chemokine functions as a chemoattractant for leukocytes and recruits monocytes, neutrophils and other effector cells from the blood to sites of infection or tissue damage. In some embodiments, the inflammatory chemokine actively participates in the inflammatory response attracting immune cells to the site of inflammation. In some embodiments, the inflammatory chemokine activates cells to initiate an immune response or promote wound healing. In some embodiments, the inflammatory chemokine is released by many different cell types and serves to guide cells of both innate immune system and adaptive immune system. Exemplary inflammatory chemokines include, but are not limited to CXCL8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10.

[0260] As used herein, “CXC chemokines,” also known as “a-chemokines,” refer to the chemokines that contain the two N-terminal cysteines separated by one amino acid, as represented in the name with an “X.” In some embodiments, the CXC chemokine is one of 17 different CXC chemokines described in mammals. In some embodiments, the CXC chemokine belongs to the category with a specific amino acid sequence (or motif) of glutamic acid-leucine- arginine (or ELR for short) immediately before the first cysteine of the CXC motif (ELR- positive). In some embodiments, the CXC chemokine belongs to the category without an ELR motif (ELR-negative). In some embodiments, the ELR-positive CXC chemokine specifically induces the migration of neutrophils, and interacts with chemokine receptors CXCR1 and CXCR2. The examples of the ELR-positive CXC chemokine include, but are not limited to, interleukin-8 (IL-8), which, for example, induces neutrophils to leave the bloodstream and enter into the surrounding tissue. In some embodiments, the CXC chemokine that lacks the ELR motif tends to be chemoattractant for lymphocytes. The examples of the ELR-negative CXC chemokine include, but are not limited to, CXCL13. In some embodiments, CXC chemokines bind to CXC chemokine receptors, of which seven have been discovered to date, designated CXCR1-7.

[0261] As used herein, “chemokine receptors” refer to cytokine receptors found on the surface of certain cells that interact with a chemokine. In some embodiments, the chemokine receptors are G protein-coupled receptors containing 7 transmembrane domains that are found predominantly on the surface of leukocytes, making it one of the rhodopsin-like receptors. In some embodiments, the chemokine receptors are one of 19 different chemokine receptors that have been characterized and share many common structural features. In some embodiments, the chemokine receptors are composed of about 350 amino acids that are divided into a short and acidic N-terminal end, seven helical transmembrane domains with three intracellular and three extracellular hydrophilic loops, and an intracellular C-terminus containing serine and threonine residues that act as phosphorylation sites during receptor regulation. In some embodiments, the chemokine receptors comprise a short and acidic N-terminal end. In some embodiments, the chemokine receptors comprise seven helical transmembrane domains. In some embodiments, the chemokine receptors comprise seven helical transmembrane domains with three intracellular and three extracellular hydrophilic loops. In some embodiments, the chemokine receptors comprise seven helical transmembrane domains with three intracellular hydrophilic loops. In some embodiments, the chemokine receptors comprise seven helical transmembrane domains with three extracellular hydrophilic loops. In some embodiments, the chemokine receptors comprise an intracellular C-terminus. In some embodiments, the chemokine receptors comprise an intracellular C-terminus containing serine and threonine residues that act as phosphorylation sites during receptor regulation. In some embodiments, the chemokine receptors comprise the first two extracellular loops that are linked together by disulfide bonding between two conserved cysteine residues. In some embodiments, the chemokine receptors comprises the N-terminal end that binds to chemokines and is important for ligand specificity. In some embodiments, the G- proteins couple to the C-terminal end of the chemokine receptor, which is important for receptor signaling following ligand binding. In some embodiments, the chemokine receptors that share high amino acid identity in their primary sequences bind different ligands. In some embodiments, more than one chemokine bind to a single receptor and the chemokine receptors are redundant in their function.

[0262] In some embodiments, the chemokine receptor is one of 20 distinct chemokine receptors discovered in humans. In some embodiments, the chemokine receptor has a rhodopsin-like 7- transmembrane structure and couples to G-protein for signal transduction within a cell. In some embodiments, the chemokine receptor is a member of a large protein family of G protein- coupled receptors. In some embodiments, following interaction with their specific chemokine ligands, the chemokine receptors trigger a flux in intracellular calcium ions (i.e., calcium signaling), which causes cell responses, including the onset of chemotaxis that traffics the cell to a desired location within the organism.

[0263] In some embodiments, the chemokine receptor belongs to one of the following families, CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors and XC chemokine receptors that correspond to the 4 distinct subfamilies of chemokines that they bind. In some embodiments, four families of chemokine receptors differ in spacing of cysteine residues near N-terminal of the receptor. In some embodiments, CXC chemokine receptors are integral membrane proteins that specifically bind and respond to cytokines of the CXC chemokine family. In some embodiments, CXC chemokine receptors represent one subfamily of chemokine receptors, a large family of G protein-linked receptors that are known as seven transmembrane (7-TM) proteins, as they span the cell membrane seven times. In some embodiments, the CXC chemokine receptors is one of six known CXC chemokine receptors in mammals, named CXCR1 through CXCR6.

[0264] As used herein, “CXCR6,” also known as C-X-C chemokine receptor type 6, BONZO, CD 186, STRL33, TYMSTR, and C-X-C motif chemokine receptor 6, refers to a chemokine receptor that is named based on its chromosomal location (within the chemokine receptor cluster on human chromosome 3p21) and its similarity to other chemokine receptors in its gene sequence. In some embodiments, CXCR6 binds the ligand CXCL16. In some embodiments, CXCR6 is more closely related in structure to CC chemokine receptors than to other CXC chemokine receptors. “CXCR6,” as used herein, includes any of the recombinant or naturally- occurring forms of CXCR6 or variants or homologs thereof that have or maintain CXCR6 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CXCR6. In some embodiments, CXCR6 is substantially identical to the protein identified by the UniProt reference number 000574 or a variant or homolog having substantial identity thereto. [0265] As used herein, “CXCL16,” also known as Chemokine (C-X-C motif) ligand 16, SCYB16, SR-PSOX, and CXCLG16, refers to a small cytokine that belongs to the CXC chemokine family. In some embodiments, CXCL16 is composed of a CXC chemokine domain, a mucin-like stalk, a transmembrane domain and a cytoplasmic tail containing a potential tyrosine phosphorylation site that may bind SH2. “CXCL16,” as used herein, includes any of the recombinant or naturally-occurring forms of CXCL16 or variants or homologs thereof that have or maintain CXCL16 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CXCL16. In some embodiments, CXCL16 is substantially identical to the protein identified by the UniProt reference number Q9H2A7 or a variant or homolog having substantial identity thereto.

[0266] As used herein, the term “fusion protein” relates to a protein which is made of polypeptide parts from different sources. Accordingly, in some embodiments, it may be also understood as a chimeric protein. In the context of the TGFBr2 fusion proteins as described herein, the term “fusion protein” is used interchangeably with the term “switch polypeptide” or “switch-receptor.” Usually, fusion proteins are proteins created through the joining of two or more genes (or, for example, cDNAs) that originally coded for separate proteins. Translation of this fusion gene (or, for example, fusion cDNA) results in a single polypeptide, for example, with functional properties derived from each of the original proteins. In some embodiments, recombinant fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics. Further details to the exemplary production of the fusion protein of the present invention are described herein.

[0267] The term “TGFBr2 switch polypeptide,” “TGFBr2 fusion protein,” or “TGFBr2 switch receptor,” as used herein, refers to the TGFBr2 fusion proteins as described herein that receive an inhibitory signal by binding to, e.g., TGF-beta, and transform (e.g., “switch”) the signal via the co-stimulatory domain of the fusion protein into an activating signal.

[0268] 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), P-galactosidase (P-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.

[0269] The term “TGFBr2” or “transforming growth factor beta receptor II,” also known as transforming growth factor, beta receptor II, TGF beta receptor 2, TGFBR2, TGFBRII, AAT3, FAA3, LDS1B, LDS2, LDS2B, MFS2, RIIC, TAAD2, TGFR-2, TGFbeta-RII, transforming growth factor beta receptor 2, TBR-ii, TBRII, refers to a protein that is a member of the serine/threonine protein kinase family and the TGFB receptor subfamily. TGFBr2 refers to a transmembrane protein that has a protein kinase domain, forms a heterodimeric complex with another receptor protein, and binds TGF-beta. TGFBr2, as used herein, includes any of the recombinant or naturally-occurring forms of TGFBr2 or variants or homologs thereof that have or maintain TGFBr2 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring TGFBr2. In some embodiments, TGFBr2 is substantially identical to the protein identified by the UniProt reference number P37173 or a variant or homolog having substantial identity thereto.

[0270] The term “GMCSFRa,” also known as CSF2RA, CD116, Cluster of Differentiation 116, CDwl l6, CSF2R, CSF2RAX, CSF2RAY, CSF2RX, CSF2RY, GM-CSF-R-alpha, GMCSFR, GMR, SMDP4, colony stimulating factor 2 receptor alpha subunit, alphaGMR, colony stimulating factor 2 receptor subunit alpha, GMR-alpha, GMCSFR-alpha, granulocytemacrophage colony-stimulating factor receptor, as used herein, refers to a receptor for granulocyte-macrophage colony-stimulating factor, which stimulates the production of white blood cells. In some embodiments, GM-CSF and its receptor play a role in earlier stages of development. In some embodiments, GMCSFRa is associated with Surfactant metabolism dysfunction type 4. GMCSFRa, as used herein, includes any of the recombinant or naturally- occurring forms of GMCSFRa or variants or homologs thereof that have or maintain GMCSFRa activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring GMCSFRa. In some embodiments, GMCSFRa is substantially identical to the protein identified by the UniProt reference number P15509 or a variant or homolog having substantial identity thereto.

[0271] The term “CD28,” also known as Cluster of Differentiation 28, CD28, Tp44, and CD28 molecule, as used herein, refers to a protein expressed on T cells that provides co-stimulatory signals required for T cell activation and survival. In some embodiments, CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins. “CD28,” as used herein, includes any of the recombinant or naturally-occurring forms of CD28 or variants or homologs thereof that have or maintain CD28 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD28. In some embodiments, CD28 is substantially identical to the protein identified by the UniProt reference number Pl 0747 or a variant or homolog having substantial identity thereto.

[0272] The term “2A” “2A self-cleaving peptide,” or “2A peptide,” as used herein, refers to a class of peptides, which can induce ribosomal skipping during translation of a protein in a cell. These peptides share a core sequence motif of DxExNPGP, and are found in a wide range of viral families. Exemplary members of 2A include, but are not limited to, P2A, E2A, F2A, and T2A. “T2A” refers to the 2A derived from thosea asigna virus, and the sequence is EGRGSLLTCGDVEENPGP (SEQ ID NO:23). “P2A” refers to the 2A derived from porcine teschovirus-1 2A, and the sequence is ATNFSLLKQAGDVEENPGP (SEQ ID NO:269). “E2A” refers to the 2 A derived from quine rhinitis A virus, and the sequence is QCTNYALLKLAGDVESNPGP (SEQ ID NO:280). F2A is derived from foot-and-mouth disease virus 18, and the sequence is VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:281). In some embodiments, adding the 1 linker “GSG” (Gly-Ser-Gly) on the N-terminal of a 2A peptide helps with efficiency.

[0273] The term “furin cleavage site,” as used herein, refers to a cleavage site recognized by protease enzyme furin, also known as FUR, PACE, PCSK3, SPC1, and paired basic amino acid cleaving enzyme. In some embodiments, furin is a subtili sin-like proprotein convertase family. In some embodiments, furin cleaves proteins just downstream of a basic amino acid target sequence (canonically, Arg-X-(Arg/Lys) -Arg'). [0274] As used herein, a “T cell receptor (TCR) fusion protein” or “TFP,” as used herein, 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.

[0275] The term “stimulation,” as used herein, refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.

[0276] The term “stimulatory molecule” or “stimulatory domain,” as used herein, refers to a molecule or portion thereof expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway. In some embodiments, the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or “ITAM”. Examples of an IT AM 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.

[0277] The term “antigen presenting cell” or “APC,” as used herein, refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC’s) on its surface. T cells may recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0278] “Major histocompatibility complex (MHC) molecules,” as used herein, are typically bound by TCRs as part of peptide:MHC complex. The MHC molecule may be an MHC class I or II molecule. The complex may be on the surface of an antigen presenting cell, such as a dendritic cell or a B cell, or any other cell, including cancer cells, or it may be immobilized by, for example, coating on to a bead or plate. [0279] The “human leukocyte antigen system (HLA),” as used herein, refers to the gene complex which encodes major histocompatibility complex (MHC) in humans and includes HLA class I antigens (A, B & C) and HLA class II antigens (DP, DQ, & DR). HLA alleles A, B and C present peptides derived mainly from intracellular proteins, e.g., proteins expressed within the cell.

[0280] During T cell development in vivo, T cells undergo a positive selection step to ensure recognition of self MHCs followed by a negative step to remove T cells that bind too strongly to MHC which present self-antigens. As a consequence, certain T cells and the TCRs they express will only recognize peptides presented by certain types of MHC molecules - i.e., those encoded by particular HLA alleles. This is known as HLA restriction.

[0281] The term “intracellular signaling domain,” as used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the TFP containing cell, e.g., a modified T-T cell. Examples of immune effector function, e.g., in a modified T-T cell, include, but are not limited to, cytolytic activity and T helper cell activity, including the secretion of cytokines. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain.

Exemplary primary intracellular signaling domains include, but are not limited to, those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In some embodiments, the intracellular signaling domain comprises a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include, but are not limited to, those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.

[0282] In some embodiment, a primary intracellular signaling domain comprises an IT AM (“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 DAP10 and DAP12.

[0283] The term “costimulatory molecule,” as used herein, refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. In some embodiments, costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Exemplary costimulatory molecules include, but are not limited to, an MHC class 1 molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD2, CD27, CD28, CD5, ICAM-1, LFA-1 (CD1 la/CD18), 4-1BB (CD137), IL-15Ra, IL12R, IL18R, IL21R, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, and FcyRIII. In some embodiments, a costimulatory intracellular signaling domain is the intracellular portion of a costimulatory molecule. In some embodiments, a costimulatory molecule is represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, IL-15Ra, IL12R, IL18R, IL21R, CD27, CD5, ICAM- 1, CD7, CD226, FcyRI, FcyRII, FcyRIII, and the like. In some embodiments, the intracellular signaling domain comprises the entire intracellular portion or the entire native intracellular signaling domain of the molecule from which it is derived, or a functional fragment thereof. [0284] The term “4-1BB,” as used herein, refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g, mouse, rodent, monkey, ape and the like; and a “4- IBB costimulatory domain,” as used herein, refers to amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g, mouse, rodent, monkey, ape and the like. “4-1BB,” also known as TNFRSF9, 4-1BB, CD137, Cluster of Differentiation 137, CDwl37, ILA, tumor necrosis factor receptor superfamily member 9, and TNF receptor superfamily member 9, as used herein, includes any of the recombinant or naturally-occurring forms of 4-1BB or variants or homologs thereof that have or maintain 4- 1BB activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring 4-1BB. In some embodiments, 4- IBB is substantially identical to the protein identified by the UniProt reference number Q07011 or a variant or homolog having substantial identity thereto.

[0285] 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. [0286] The terms “antibody fragment,” as used herein, 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 multispecific antibodies formed from antibody fragments.

[0287] The term “scFv,” as used herein, 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.

[0288] The term “heavy chain variable region” or “VH” with regard to an antibody, as used herein, 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, as used herein, refers to a heavy chain comprising a single variable antibody domain. [0289] 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. In some embodiments, the scFv may comprise -linker-Vu or may comprise Vu-linker- .

[0290] In some embodiments, 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), or a single chain antibody (scFv) derived from a murine, humanized or human antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). 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.

[0291] The term “recombinant antibody,” as used herein, 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.

[0292] The term “antigen” or “Ag,” as used herein, refers to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. In some embodiments, this immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.

[0293] 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.

[0294] The term “recombinant nucleic acid” and the term “recombinant nucleic acid molecule” are used interchangeably.

[0295] The term “CD3” or “Cluster of Differentiation 3,” as used herein, refers to a protein complex that is part of the T cell receptor that is involved in activating both the cytotoxic T cell and T helper cells. In some embodiments, it is composed of four distinct chains. For example, in some embodiments, the complex contains a CD3y chain, a CD36 chain, and two CD3s chains in mammals.

[0296] “ CD3s,” “CD3s chain,” or “T-cell surface glycoprotein CD3 epsilon chain,” as used herein, includes any of the recombinant or naturally-occurring forms of CD3s or variants or homologs thereof that have or maintain CD3s activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD3s. In some embodiments, CD3s is substantially identical to the protein identified by the UniProt reference number P07766 or a variant or homolog having substantial identity thereto.

[0297] “CD36,” “CD36 chain,” or “T-cell surface glycoprotein CD3 delta chain,” as used herein, includes any of the recombinant or naturally-occurring forms of CD36 or variants or homologs thereof that have or maintain CD36 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD36. In some embodiments, CD36 is substantially identical to the protein identified by the UniProt reference number P04234 or a variant or homolog having substantial identity thereto.

[0298] “CD3y,” “CD3y chain,” or “T-cell surface glycoprotein CD3 gamma chain,” as used herein, includes any of the recombinant or naturally-occurring forms of CD3y or variants or homologs thereof that have or maintain CD3y activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD3y. In some embodiments, CD3y is substantially identical to the protein identified by the UniProt reference number P09693 or a variant or homolog having substantial identity thereto.

[0299] As used herein, the term “CD 19,” also known as B-lymphocyte antigen CD 19, B4, CVID3, and CD19 molecule, refers to the Cluster of Differentiation 19 protein, which is an antigenic determinant detectable on B cell leukemia precursor cells, other malignant B cells and most cells of the normal B cell lineage. CD 19, as used herein, includes any of the recombinant or naturally-occurring forms of CD 19 or variants or homologs thereof that have or maintain CD19 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD 19. In some embodiments, CD 19 is substantially identical to the protein identified by the UniProt reference number P15391 or a variant or homolog having substantial identity thereto.

[0300] As used herein, the term “BCMA” refers to the B-cell maturation antigen, also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), Cluster of Differentiation 269 protein (CD269), BCM, TNFRSF13A, tumor necrosis factor receptor superfamily member 17, and TNF receptor superfamily member 17, which is a protein that in humans is encoded by the TNFRSF17 gene. TNFRSF17 is a cell surface receptor of the TNF receptor superfamily which recognizes B-cell activating factor (BAFF) (see, e.g., Laabi et al., EMBO 11 (11): 3897- 904 (1992). This receptor is expressed in mature B lymphocytes, and may be important for B- cell development and autoimmune response. BCMA, as used herein, includes any of the recombinant or naturally-occurring forms of BCMA or variants or homologs thereof that have or maintain BCMA activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring BCMA. In some embodiments, BCMA is substantially identical to the protein identified by the UniProt reference number Q02223 or a variant or homolog having substantial identity thereto.

[0301] As used herein, the term “CD 16,” also known as FcyRIII, refers to a cluster of differentiation molecule found on the surface of natural killer cells, neutrophil polymorphonuclear leukocytes, monocytes, and macrophages. CD16 has been identified as Fc receptors FcyRIIIa (CD 16a) and FcyRIIIb (CD 16b), which participate in signal transduction. In some embodiments, CD 16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC). CD 16, as used herein, includes any of the recombinant or naturally-occurring forms of CD 16 or variants or homologs thereof that have or maintain CD 16 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD 16. In some embodiments, CD 16 is substantially identical to the protein identified by the UniProt reference number P08637 (CD16a) or a variant or homolog having substantial identity thereto or the protein identified by the UniProt reference number 07501 (CD16b) or a variant or homolog having substantial identity thereto.

[0302] As used herein, the term “NKG2D,” also known as KLRK1, CD314, D12S2489E, KLR, NKG2-D, NKG2D, natural killer group 2D, killer cell lectin-like receptor KI, and killer cell lectin like receptor KI, refers to a transmembrane protein belonging to the CD94/NKG2 family of C-type lectin-like receptors. In some embodiments, in humans, NKG2D is expressed by NK cells, y6 T cells and CD8+ aP T cells. In some embodiments, NKG2D recognizes induced-self proteins from MIC and RAET1/ULBP families which appear on the surface of stressed, malignant transformed, and infected cells. NKG2D, as used herein, includes any of the recombinant or naturally-occurring forms of NKG2D or variants or homologs thereof that have or maintain NKG2D activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring NKG2D. In some embodiments, NKG2D is substantially identical to the protein identified by the UniProt reference number P26718 or a variant or homolog having substantial identity thereto.

[0303] The term “mesothelin (MSLN),” also known as MPF and SMRP, refers to a tumor differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. In some embodiments, mesothelin is over-expressed in several human tumors, including mesothelioma and ovarian and pancreatic adenocarcinoma. MSLN, as used herein, includes any of the recombinant or naturally-occurring forms of MSLN or variants or homologs thereof that have or maintain MSLN activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring MSLN. In some embodiments, MSLN is substantially identical to the protein identified by the UniProt reference number QI 3421 or a variant or homolog having substantial identity thereto.

[0304] The term “tyrosine-protein kinase transmembrane receptor ROR1”, also known as ROR1, neurotrophic tyrosine kinase, receptor-related 1 (NTRKR1), dJ537F10.1, receptor tyrosine kinase-like orphan receptor 1, and receptor tyrosine kinase like orphan receptor 1, refers to a member of the receptor tyrosine kinase-like orphan receptor (ROR) family. It plays a role in metastasis of cancer. R0R1, as used herein, includes any of the recombinant or naturally- occurring forms of R0R1 or variants or homologs thereof that have or maintain R0R1 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring ROR1. In some embodiments, ROR1 is substantially identical to the protein identified by the UniProt reference number Q01973 or a variant or homolog having substantial identity thereto.

[0305] The term “MUC16”, also known as mucin 16, cell-surface associated, ovarian cancer- related tumor marker CA125, CA-125 (cancer antigen 125, carcinoma antigen 125, or carbohydrate antigen 125), mucin 16, and CA125, refers to a membrane-tethered mucin that contains an extracellular domain at its amino terminus, a large tandem repeat domain, and a transmembrane domain with a short cytoplasmic domain. In some embodiments, products of this gene have been used as a marker for different cancers, with higher expression levels associated with poorer outcomes. MUC16, as used herein, includes any of the recombinant or naturally-occurring forms of MUC16 or variants or homologs thereof that have or maintain MUC16 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring MUC16. In some embodiments, MUC16 is substantially identical to the protein identified by the UniProt reference number Q8WXI7 or a variant or homolog having substantial identity thereto.

[0306] The term “CD22,” also known as cluster of differentiation-22, sialic acid binding Ig-like lectin 2, SIGLEC-2, SIGLEC2, CD22 molecule, T cell surface antigen leu- 14, and B cell receptor CD22, refers to a protein that mediates B cell/B cell interactions, and is thought to be involved in, e.g., the localization of B cells in lymphoid tissues. In some embodiments, CD22 is associated with diseases including, but not limited to, refractory hematologic cancer and hairy cell leukemia. An exemplary fully human anti-CD22 monoclonal antibody (“M971”) suitable for use with the methods as described herein is described, e.g., in Xiao et al., MAbs. 2009 May- Jun; 1(3): 297-303. CD22, as used herein, includes any of the recombinant or naturally- occurring forms of CD22 or variants or homologs thereof that have or maintain CD22 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD22. In some embodiments, CD22 is substantially identical to the protein identified by the UniProt reference number P20273 or a variant or homolog having substantial identity thereto.

[0307] “Programmed cell death protein 1,” also known as PD-1, CD279 (cluster of differentiation 279), PDCD1, PD1, SLEB2, hPD-1, hSLEl, and Programmed cell death 1, refers to a protein on the surface of cells that has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting selftolerance by suppressing T cell inflammatory activity. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells. PD-1 is an immune checkpoint and guards against autoimmunity, e.g., through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T-cells in lymph nodes. Second, it reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells). PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, PD-L1 and PD-L2. PD-1, as used herein, includes any of the recombinant or naturally-occurring forms of PD-1 or variants or homologs thereof that have or maintain PD-1 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PD-1. In some embodiments, PD-1 is substantially identical to the protein identified by the UniProt reference number Q15116 or a variant or homolog having substantial identity thereto.

[0308] “Programmed death-ligand 1 (PD-L1),” also known as cluster of differentiation 274, CD274, B7 homolog 1, B7-H, B7-H1, B7H1, PDCD1L1, PDCD1LG1, PDL1, hPD-Ll, and CD274 molecule, refers to a 40kDa type 1 transmembrane protein. In some embodiments, PD- L1 may play a major role in suppressing the adaptive arm of immune system during particular events such as, e.g., pregnancy, tissue allografts, autoimmune disease and other disease states such as, e.g., hepatitis. Normally the adaptive immune system reacts to antigens that are associated with immune system activation by exogenous or endogenous danger signals. In turn, clonal expansion of antigen-specific CD8+ T cells and/or CD4+ helper cells is propagated. The binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via Immunoreceptor Tyrosine-Based Switch Motif (ITSM) motif. This reduces the proliferation of antigen-specific T-cells in lymph nodes, while simultaneously reducing apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells) - further mediated by a lower regulation of the gene Bcl-2. PD-L1, as used herein, includes any of the recombinant or naturally-occurring forms of PD-L1 or variants or homologs thereof that have or maintain PD-L1 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PD-L1. In some embodiments, PD-L1 is substantially identical to the protein identified by the UniProt reference number Q9NZQ7 or a variant or homolog having substantial identity thereto.

[0309] The term “PD-L2”, also known as B7-DC, is a ligand of PD-1. The amino acid sequence of full length PD-L2 is provided in the Gene Bank under accession number NP 079515.2. The term “PD-L1” also includes protein variants of PD-L1. The term “PD-L2” includes recombinant PD-L2 or fragments thereof. The term also includes, for example, affinity tagged (e.g., histidine tagged) PD-L2 or fragments thereof, mouse or human Fc tagged PD-L2 or fragments thereof, or PD-L2 or fragments thereof coupled to a signal sequence, for example, ROR1.

[0310] PD- L2, as referred herein, includes any of the recombinant or naturally-occurring forms of PD- L2 or variants or homologs thereof that have or maintain PD-L2 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PD-L2. In some embodiments, PD-L2 is substantially identical to the protein identified by the UniProt reference number Q9BQ51 or a variant or homolog having substantial identity thereto.

[0311] The “CD79a (Cluster of Differentiation 79a)” and “CD79P (Cluster of Differentiation 79P)” genes encode proteins that make up the B lymphocyte antigen receptor, a multimeric complex that includes the antigen-specific component, surface immunoglobulin (Ig). Surface Ig non-covalently associates with two other proteins, Ig-alpha and Ig-beta (encoded by CD79a and its paralog CD79P, respectively) which are necessary for expression and function of the B-cell antigen receptor. Functional disruption of this complex can lead to, e.g., human B-cell chronic lymphocytic leukemias. CD79a protein, as used herein, includes any of the recombinant or naturally-occurring forms of CD79a protein or variants or homologs thereof that have or maintain CD79a protein activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD79a protein. In some embodiments, CD79a protein is substantially identical to the protein identified by the UniProt reference number Pl 1912 or a variant or homolog having substantial identity thereto. CD79P protein, as used herein, includes any of the recombinant or naturally-occurring forms of CD79P protein or variants or homologs thereof that have or maintain CD79P protein activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD79P protein. In some embodiments, CD79P protein is substantially identical to the protein identified by the UniProt reference number P40259 or a variant or homolog having substantial identity thereto.

[0312] “ CD70,” also known as CD27LG and TNFSF7, as referred herein, refers to a cytokine that is the ligand for CD27. The CD70-CD27 pathway plays an important role in the generation and maintenance of T cell immunity, in particular, during antiviral responses. Upon CD27 binding, CD70 induces the proliferation of co-stimulated T-cells and enhances the generation of cytolytic T-cells. CD70, as referred herein, includes any of the recombinant or naturally- occurring forms of CD70 or variants or homologs thereof that have or maintain CD70 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD70. In some embodiments, CD70 is substantially identical to the protein identified by the UniProt reference number P32970 or a variant or homolog having substantial identity thereto.

[0313] “Prostate-specific membrane antigen” (PSMA), also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I), NAAG peptidase, FOLH1, FGCP, FOLH, GCP2, GCPII, NAALAD1, NAALAdase, PSM, mGCP, folate hydrolase (prostate-specific membrane antigen) 1, or folate hydrolase 1 is a type II membrane protein expressed in all forms of prostate tissue, including carcinoma. The PSMA protein has a unique 3-part structure: a 19-amino-acid internal portion, a 24-amino-acid transmembrane portion, and a 707-amino-acid external portion. PSMA acts as a glutamate- preferring carboxypeptidase. PMSA expression is increased in cancer tissue in the prostate. PSMA, as referred herein, includes any of the recombinant or naturally-occurring forms of PSMA or variants or homologs thereof that have or maintain PSMA activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PSMA. In some embodiments, PSMA is substantially identical to the protein identified by the UniProt reference number Q04609 or a variant or homolog having substantial identity thereto.

[0314] “HER2,” also known as receptor tyrosine-protein kinase erbB-2, CD340 (cluster of differentiation 340), proto-oncogene Neu, ERBB2, human epidermal growth factor receptor 2, HER2/neu, HER-2, HER-2/neu, HER2, MLN 19, NEU, NGL, TKR1, erb-b2 receptor tyrosine kinase 2, encodes a member of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases. This protein has no ligand binding domain of its own and therefore cannot bind growth factors. However, it does bind tightly to other ligand-bound EGF receptor family members to form a heterodimer, stabilizing ligand binding and enhancing kinase-mediated activation of downstream signaling pathways, such as those involving mitogen-activated protein kinase and phosphatidylinositol-3 kinase. Amplification and/or overexpression of this gene has been reported in numerous cancers, including breast and ovarian tumors. Alternative splicing results in several additional transcript variants, some encoding different isoforms and others that have not been fully characterized. HER2 is amplified and/or overexpressed in 20-30% of invasive breast carcinomas. HER2 -positive breast cancer is treated in a separate manner from other subtypes of breast cancer and commonly presents as more aggressive disease. HER2, as referred herein, includes any of the recombinant or naturally-occurring forms of HER2 or variants or homologs thereof that have or maintain HER2 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HER2. In some embodiments, HER2 is substantially identical to the protein identified by the UniProt reference number P04626 or a variant or homolog having substantial identity thereto.

[0315] The term “CD22,” also known as cluster of differentiation-22, sialic acid binding Ig-like lectin 2, SIGLEC-2, SIGLEC2, CD22 molecule, T cell surface antigen leu- 14, and B cell receptor CD22, refers to a protein that mediates B cell/B cell interactions, and is thought to be involved in, e.g., the localization of B cells in lymphoid tissues. In some embodiments, CD22 is associated with diseases including, but not limited to, refractory hematologic cancer and hairy cell leukemia. An exemplary fully human anti-CD22 monoclonal antibody (“M971”) suitable for use with the methods as described herein is described, e.g., in Xiao et al., MAbs. 2009 May- Jun; 1(3): 297-303. CD22, as referred herein, includes any of the recombinant or naturally- occurring forms of CD22 or variants or homologs thereof that have or maintain CD22 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD22. In some embodiments, CD22 is substantially identical to the protein identified by the UniProt reference number P20273 or a variant or homolog having substantial identity thereto.

[0316] The term “interleukin 15 receptor” or “IL-15R” refers to a type I cytokine receptor that IL-15 binds to and signals through. In some embodiments, IL-15R is composed of three subunits: IL- 15 receptor alpha chain (“IL-15Ra” or CD215), IL-2 receptor beta chain (“IL-2RP” or CD122) and IL-2 receptor gamma/the common gamma chain (“IL-2RY/YC” or CD132). For example, in some embodiments, 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. In some embodiments, IL-15Ra contains a Sushi domain (amino acid 31-95), which is essential for IL-15 binding. In some embodiments, IL-15Ra exists as a soluble form (sIL-15Ra). In some embodiments, 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. In some embodiments, the human sIL- 15Ra, about 42 kDa in size, may prolong the half-life of IL-15 or potentiate IL-15 signaling through IL-15 binding and IL-2RP/YC heterodimer. Although IL-15R shares subunits with IL-2R that contain the cytoplasmic motifs required for signal transduction, in some embodiments, 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-15Ra binding. In some embodiments, IL-15 binds to IL-15Ra specifically with high affinity, which then associates with a complex composed of IL-2RP and fL-2Ry/yc subunits, expressed on the same cell (“cis- presentation”) or on a different cell (“trans-presentation”). In some embodiments, the interaction between IL-15 and IL-15Ra is independent of the complex composed of IL-2RP and fL-2Ry/yc subunits. In some embodiments, IL- 15 binding to the IL-2Rp/yc heterodimeric receptor induces JAK1 activation that phosphorylates STAT3 via the beta chain, and JAK3 activation that phosphorylates STAT5 via the gamma chain. In some embodiments, the IL-15/IL-15R interaction modulates T-cell development and homeostasis in memory CD8+ T-cell. In some embodiments, the IL-15/IL-15R interaction also modulates NK cell development, maintenance, expansion and activities.

[0317] In some embodiments, IL-15Ra cytoplasmic (or intracellular) domain comprises amino acids 229-267 of IL-15Ra protein. In some embodiments, IL-15Ra cytoplasmic (or intracellular) domain comprises a sequence of SEQ ID NO:372. In some embodiments, IL-15Ra Sushi domain comprises amino acids 31-95 of IL-15Ra protein. In some embodiments, IL-15Ra Sushi domain comprises a sequence of SEQ ID NO:382. In some embodiments, IL-15Ra comprises the transmembrane domain and the cytoplasmic (intracellular) domain of IL-15Ra protein. In some embodiments, IL-15Ra comprises amino acids 96-267 of IL-15Ra protein. In some embodiments, IL-15Ra comprises a sequence of SEQ ID NO:383. In some embodiments, sIL-15Ra comprises amino acids 21-205 of IL-15Ra protein. In some embodiments, sIL-15Ra comprises a sequence of SEQ ID NO:379.

[0318] SEQ ID NO:372 (IL-15Ra intracellular domain) KSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL [0319] SEQ ID NO:379 (Soluble IL-15Ra (sIL-15Ra)) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGS QLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT [0320] SEQ ID NO:382 (IL-15Ra Sushi domain) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIR

[0321] SEQ ID NO:383 (IL-15Ra region downstream of Sushi domain) DPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPS KSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLL CGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL

[0322] SEQ ID NO:386 (IL-15Ra full-length protein sequence)

MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICN SGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQP ESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAK NWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVE ME AME ALPVTWGTS SRDEDLENCSHHL

[0323] SEQ ID NO:300 (IL-15Ra transmembrane domain) VAISTSTVLLCGLSAVSLLACYL

[0324] “IL-15Ra,” also known as CD215, IL-15 receptor subunit alpha, IL-15R-alpha, IL- 15RA, and Interleukin- 15 receptor subunit alpha, as used herein, includes any of the recombinant or naturally-occurring forms of IL-15Ra or variants or homologs thereof that have or maintain IL-15Ra activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL-15Ra. In some embodiments, IL-15Ra is substantially identical to the protein identified by the UniProt reference number QI 3261 or a variant or homolog having substantial identity thereto.

[0325] “IL-2RP,” also known as CD 122, IL-2 receptor subunit beta, IL-2R subunit beta, IL- 2RB, P70-75, IMD63, and Interleukin-2 receptor subunit beta, as used herein, includes any of the recombinant or naturally-occurring forms of IL-2RP or variants or homologs thereof that have or maintain IL-2RP activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL-2Rp. In some embodiments, IL-2RP is substantially identical to the protein identified by the UniProt reference number P14784 or a variant or homolog having substantial identity thereto.

[0326] “IL-2 receptor gamma/the common gamma chain,” also known as IL-2RY/YC, IL2RG, CIDX, IL-2RG, IMD4, P64, SCIDX, SCIDX1, interleukin 2 receptor subunit gamma, or CD 132, as used herein, includes any of the recombinant or naturally-occurring forms of IL- 2Ry/yc or variants or homologs thereof that have or maintain IL-2RY/YC activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL-2Ry/yc. In some embodiments, IL-2Ry/yc is substantially identical to the protein identified by the UniProt reference number P31785 or a variant or homolog having substantial identity thereto.

[0327] As used herein, the terms “cleave” or “cleavage” refer to the hydrolysis of phosphodiester bonds within the backbone of a recognition sequence within a target sequence that results in a double-stranded break within the target sequence, referred to herein as a “cleavage site”. In some embodiments, the cleavage site can be a self-cleaving peptide such as a T2A, P2A, E2A or F2A cleavage site. In some embodiments, the cleavage site can comprise a sequence of SEQ ID NO: 1261 (P2A: GSGATNFSLLKQAGDVEENPG).

[0328] The term “transfer vector,” as used herein, refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “transfer vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0329] The term “expression vector,” as used herein, refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0330] The term “lentivirus,” as used herein, refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.

[0331] The term “lentiviral vector,” as used herein, refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR™ gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen, and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0332] The term “homologous” or “identity,” as used herein, refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g, 9 of 10), are matched or homologous, the two sequences are 90% homologous.

[0333] “Humanized” forms of non-human (e.g, murine) antibodies, as described herein, are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antib ody/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.

[0334] “Human” or “fully human,” as used herein, refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.

[0335] The term “isolated,” as used herein, means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0336] 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.

[0337] The term “conservative sequence modifications,” as used herein, 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. [0338] The term “operably linked” or “transcriptional control,” as used herein, refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. Alternatively, in some embodiments, the term “operably linked,” as used herein, refers to functional linkage between two heterologous nucleic acid sequence resulting in expression of both. 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.

[0339] The terms “nucleotide,” “nucleic acid” and “polynucleotide,” as used herein, are used interchangeably, and refer to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);

Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0340] The terms “peptide,” “polypeptide,” and “protein,” as used herein, are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0341] The term “promoter,” as used herein, refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0342] The term “promoter/regulatory sequence,” as used herein, refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some embodiments, 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.

[0343] The term “constitutive” promoter, as used herein, refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. [0344] The term “inducible” promoter, as used herein, refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

[0345] The term “tissue-specific” promoter, as used herein, refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0346] 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 (GlysSer). Also included within the scope of the present disclosure are linkers described in WO2012/138475 (incorporated herein by reference). In some embodiments, the linker sequence comprises (G4S)_n, wherein n=2 to 5. In some embodiments, the linker sequence comprises (G4S)_n, wherein n=l to 3.

[0347] 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.

[0348] As used herein, “/// 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.

[0349] As used herein, a “poly(A)” refers to a series of adenosines attached by polyadenylation to the mRNA. In the preferred embodiment of a construct for transient expression, the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.

[0350] 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 poly adenylation signal. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3’ end at the cleavage site.

[0351] 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.

[0352] The term “signal transduction pathway,” as used herein, refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.

[0353] The term, a “substantially purified” cell, as used herein, refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some embodiments, 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.

[0354] 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)).

[0355] The term “transfected” or “transformed” or “transduced,” as used herein, refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0356] The term “specifically binds,” as used herein, refers to an antibody, an antibody fragment or a specific ligand, which recognizes and binds a cognate binding partner (e.g., CD 19) present in a sample, but which does not necessarily and substantially recognize or bind other molecules in the sample.

[0357] 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.

[0358] The term “anti-tumor effect,” as used herein, 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. In some embodiments, 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.

[0359] The term “autologous,” as used herein, refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0360] The term “allogeneic” or, alternatively, “allogenic,” as used herein, 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 antigenicallys.

[0361] The term “xenogeneic,” as used herein, refers to a graft derived from an animal of a different species.

[0362] The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein. The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is a cancer. In some aspects, the tumor is a solid tumor. In some aspects, the tumor is a hematologic malignancy.

[0363] The term “cancer,” as used herein, 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.

[0364] The term “cytotoxic agent,” as used herein, refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.

[0365] A “chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer. Chemotherapeutic agents include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer.

[0366] The term “encoding,” as used herein, 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 noncoding 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.

[0367] 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. [0368] The terms “effective amount” and “therapeutically effective amount,” as used herein, 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. [0369] The term “endogenous,” as used herein, refers to any material from or produced inside an organism, cell, tissue or system.

[0370] The term “exogenous,” as used herein, refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0371] The term “expression,” as used herein, refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

[0372] The term “parenteral” administration of an immunogenic composition, as used herein, includes, e.g, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, intratumoral, or infusion techniques.

[0373] The term “therapeutic” as used herein, means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.

[0374] The term “prophylaxis” as used herein, means the prevention of or protective treatment for a disease or disease state.

[0375] The term “functional disruption,” as used herein, refers to a physical or biochemical change to a specific (e.g., target) nucleic acid (e.g., gene, RNA transcript, of protein encoded thereby) that prevents its normal expression and/or behavior in the cell. In one embodiment, a functional disruption refers to a modification of the gene via a gene editing method. In one embodiment, a functional disruption prevents expression of a target gene (e.g, an endogenous gene).

[0376] As used herein, the term “meganuclease” refers to an endonuclease that binds doublestranded DNA at a recognition sequence that is greater than 12 base pairs. In some embodiments, the recognition sequence for a meganuclease of the present disclosure is 22 base pairs. In some embodiments, a meganuclease may be an endonuclease that is derived from I- Crel and may refer to an engineered variant of I-Crel that has been modified relative to natural I- Crel with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA- binding affinity, or dimerization properties. Methods for producing such modified variants of I- Crel are known in the art (e.g., WO 2007/047859). In some embodiments, a meganuclease binds to double-stranded DNA as a heterodimer or as a “single-chain meganuclease” in which a pair of DNA-binding domains are joined into a single polypeptide using a peptide linker. The term “homing endonuclease” is synonymous with the term “meganuclease.” In some embodiments, meganucleases are substantially non-toxic when expressed in cells, particularly in human T cells, such that cells may be transfected and maintained at 37°C without observing deleterious effects on cell viability or significant reductions in meganuclease cleavage activity when measured using the methods described herein.

[0377] As used herein, the term “single-chain meganuclease” refers to a polypeptide comprising a pair of nuclease subunits joined by a linker. A single-chain meganuclease has the organization: N-terminal subunit - Linker - C-terminal subunit. In some embodiments, the two meganuclease subunits may generally be non-identical in amino acid sequence and may recognize nonidentical DNA sequences. Thus, in some embodiments, single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences. In some embodiments, a single-chain meganuclease may be referred to as a “single-chain heterodimer” or “single-chain heterodimeric meganuclease” although it is not, in fact, dimeric. For clarity, unless otherwise specified, the term “meganuclease” can refer to a dimeric or single-chain meganuclease.

[0378] 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.

[0379] 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.

[0380] 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.

[0381] As used herein, the term “megaTAL” refers to a single-chain nuclease comprising a transcription activator-like effector (TALE) DNA binding domain with an engineered, sequencespecific homing endonuclease.

[0382] As is used herein, the terms “T cell receptor” and “T cell receptor complex” are used interchangeably to refer to a molecule found on the surface of T cells that is, in general, responsible for recognizing antigens. The TCR comprises a heterodimer consisting of a TCR alpha and TCR beta chain in 95% of T cells, whereas 5% of T cells have TCRs consisting of TCR gamma and TCR delta chains. The TCR further comprises one or more of CD3s, CD3y, and CD36. In some embodiments, the TCR comprises CD3s. In some embodiments, the TCR comprises CD3y. In some embodiments, the TCR comprises CD35. In some embodiments, the TCR comprises CD3(^. Engagement of the TCR with antigen, e.g., with antigen and MHC, results in activation of its T cells through a series of biochemical events mediated by associated enzymes, co-receptors, and specialized accessory molecules. In some embodiments, the constant domain of human TCR alpha has a sequence of SEQ ID NO: 142. In some embodiments, the constant domain of human TCR alpha has an IgC domain having a sequence of SEQ ID NO: 143, a transmembrane domain having a sequence of SEQ ID NO: 144, and an intracellular domain having a sequence of SS. In some embodiments, the constant domain of murine TCR alpha has a sequence of SEQ ID NO: 147. In some embodiments, the constant domain of murine TCR alpha has a transmembrane domain having a sequence of SEQ ID NO: 144, and an intracellular domain having a sequence of SS. In some embodiments, the constant domain of human TCR beta has a sequence of SEQ ID NO: 148. In some embodiments, the constant domain of human TCR beta has an IgC domain having a sequence of SEQ ID NO: 149, a transmembrane domain having a sequence of SEQ ID NO: 150, and an intracellular domain having a sequence of SEQ ID NO: 151. In some embodiments, the constant domain of murine TCR beta has a sequence of SEQ ID NO: 152. In some embodiments, the constant domain of murine TCR beta has a transmembrane domain having a sequence of SEQ ID NO: 152, and an intracellular domain having a sequence of SEQ ID NO: 153. In some embodiments, the constant domain of human TCR delta has a sequence of SEQ ID NO:243. In some embodiments, the constant domain of human TCR delta has an IgC domain having a sequence of SEQ ID NO:265, a transmembrane domain having a sequence of SEQ ID NO: 158, and an intracellular domain having a sequence of L. In some embodiments, the constant domain of human TCR gamma has a sequence of SEQ ID NO:21. In some embodiments, the constant domain of human TCR gamma has an IgC domain having a sequence of SEQ ID NO: 155, a transmembrane domain having a sequence of SEQ ID NO: 156, and an intracellular domain having a sequence of SEQ ID NO: 157.

[0383] In some embodiments, human CD3 epsilon has a sequence of SEQ ID NO:258. In some embodiments, human CD3 epsilon has an extracellular domain having a sequence of SEQ ID NO: 126, a transmembrane domain having a sequence of SEQ ID NO: 127, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO: 128. In some embodiments, human CD3 delta has a sequence of SEQ ID NO: 136. In some embodiments, human CD3 delta has an extracellular domain having a sequence of SEQ ID NO: 138, a transmembrane domain having a sequence of SEQ ID NO: 139, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO: 140. In some embodiments, human CD3 gamma has a sequence of SEQ ID NO: 130. In some embodiments, human CD3 gamma has an extracellular domain having a sequence of SEQ ID NO: 132, a transmembrane domain having a sequence of SEQ ID NO: 133, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO: 134. [0384] 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.

[0385] Provided herein are compositions of matter and methods of use for the treatment of a disease such as cancer, using recombinant nucleic acids comprising: a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP), wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked, and a second nucleic acid sequence encoding CXCR6 or a functional fragment there. 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 colocated 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 costimulatory molecule such as 4-1BB (i.e., CD137), CD27 and/or CD28.

C-X-C chemokine receptor type 6 (CXCR6) [0386] Provided herein are recombinant nucleic acids comprising: a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked, and a second nucleic acid sequence encoding C-X-C chemokine receptor type 6 (CXCR6) or a functional fragment thereof. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a linker. In some embodiments, the linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A cleavage site. In some embodiments, the cleavage site can be a self-cleaving peptide such as a T2A, P2A, E2A or F2A cleavage site. In some embodiments, the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.

[0387] In some embodiments, CXCR6 or a functional fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence listed in Table 4 or a fragment thereof. In some embodiments, CXCR6 or a functional fragment thereof comprises any one sequence listed in Table 4 or a fragment thereof. In some embodiments, the sequence of CXCR6 or a functional fragment thereof is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to any one sequence listed in Table 4 or a fragment thereof. In some embodiments, the sequence of CXCR6 or a functional fragment thereof is any one sequence listed in Table 4 or a fragment thereof.

[0388] In some embodiments, CXCR6 or a functional fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID N0s:400-402. In some embodiments, CXCR6 or a functional fragment thereof comprises any one sequence selected from SEQ ID N0s:400-402. In some embodiments, the sequence of CXCR6 or a functional fragment thereof is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to any one sequence selected from SEQ ID N0s:400-402. In some embodiments, the sequence of CXCR6 or a functional fragment thereof is any one sequence selected from SEQ ID N0s:400-402.

[0389] In some embodiments, CXCR6 or a functional fragment thereof comprises an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of CXCR6 or a functional fragment thereof as described herein. For example, in some embodiments, CXCR6 or a functional fragment thereof comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of a sequence of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of any one sequence selected from SEQ ID NO:400 - SEQ ID NO:402. In some embodiments, CXCR6 or a functional fragment thereof comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of any one sequence selected from SEQ ID NO:400 - SEQ ID NO:402.

[0390] In some embodiments, CXCR6 or a functional fragment thereof comprises amino acid residue deletions from the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of CXCR6 or a functional fragment thereof as described herein. For example, in some embodiments, CXCR6 or a functional fragment thereof comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C-terminal end of a sequence of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C-terminal end of a sequence of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C-terminal end of any one sequence selected from SEQ ID NO:400 - SEQ ID NO:402. In some embodiments, CXCR6 or a functional fragment thereof comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C-terminal end of any one sequence selected from SEQ ID NO:400 - SEQ ID NO:402. In some embodiments, CXCR6 or a functional fragment thereof comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of a sequence of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of a sequence of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of any one sequence selected SEQ ID NO:400 - SEQ ID NO:402. In some embodiments, CXCR6 or a functional fragment thereof comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of any one sequence selected from SEQ ID NO:400 - SEQ ID NO:402.

[0391] In some embodiments, the sequence of the recombinant nucleic acid encoding CXCR6 or a functional fragment thereof is codon optimized. In some embodiments, the CXCR6 or functional fragment thereof is encoded by a nucleic acid with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 76%, 96%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO:427. In some embodiments, the CXCR6 or functional fragment thereof is encoded by a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:427. In some embodiments, the CXCR6 or functional fragment thereof is encoded by the nucleic acid sequence of SEQ ID NO:427.

[0392] Human CXCR6 full sequence

MAEHDYHEDYGFSSFNDSSQEEHQDFLQFSKVFLPCMYLVVFVCGLVGNSLVLVISIFY HKLQSLTDVFLVNLPLADLVFVCTLPFWAYAGIHEWVFGQVMCKSLLGIYTINFYTSM LILTCITVDRFIVVVKATKAYNQQAKRMTWGKVTSLLIWVISLLVSLPQIIYGNVFNLDK LICGYHDEAISTVVLATQMTLGFFLPLLTMIVCYSVIIKTLLHAGGFQKHRSLKIIFLVMA VFLLTQMPFNLMKFIRSTHWEYYAMTSFHYTIMVTEAIAYLRACLNPVLYAFVSLKFR KNFWKLVKDIGCLPYLGVSHQWKSSEDNSKTFSASHNVEATSMFQL (SEQ ID NO:400) [0393] Human CXCR6 natural variant E3K sequence MAKHDYHEDYGFSSFNDSSQEEHQDFLQFSKVFLPCMYLVVFVCGLVGNSLVLVISIFY HKLQSLTDVFLVNLPLADLVFVCTLPFWAYAGIHEWVFGQVMCKSLLGIYTINFYTSM LILTCITVDRFIVVVKATKAYNQQAKRMTWGKVTSLLIWVISLLVSLPQIIYGNVFNLDK LICGYHDEAISTVVLATQMTLGFFLPLLTMIVCYSVIIKTLLHAGGFQKHRSLKIIFLVMA VFLLTQMPFNLMKFIRSTHWEYYAMTSFHYTIMVTEAIAYLRACLNPVLYAFVSLKFR KNFWKLVKDIGCLPYLGVSHQWKSSEDNSKTFSASHNVEATSMFQL (SEQ ID NO:401) [0394] Human CXCR6 variant D25 A sequence

MAEHDYHEDYGFSSFNDSSQEEHQAFLQFSKVFLPCMYLVVFVCGLVGNSLVLVISIFY HKLQSLTDVFLVNLPLADLVFVCTLPFWAYAGIHEWVFGQVMCKSLLGIYTINFYTSM LILTCITVDRFIVVVKATKAYNQQAKRMTWGKVTSLLIWVISLLVSLPQIIYGNVFNLDK LICGYHDEAISTVVLATQMTLGFFLPLLTMIVCYSVIIKTLLHAGGFQKHRSLKIIFLVMA

VFLLTQMPFNLMKFIRSTHWEYYAMTSFHYTIMVTEAIAYLRACLNPVLYAFVSLKFR

KNFWKLVKDIGCLPYLGVSHQWKSSEDNSKTFSASHNVEATSMFQL (SEQ ID NO:402)

Extracellular Domain

[0395] In some embodiments, CXCR6 or a functional fragment thereof comprises at least one extracellular domain. In some embodiments, CXCR6 or a functional fragment thereof comprises at least two extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at least three extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at least four extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at most one extracellular domain. In some embodiments, CXCR6 or a functional fragment thereof comprises at most two extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at most three extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at most four extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises one extracellular domain. In some embodiments, CXCR6 or a functional fragment thereof comprises two extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises three extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises four extracellular domains.

[0396] In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:403. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region comprising the sequence of SEQ ID NO:403. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:403. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region of which sequence is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to SEQ ID NO:403. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region of which sequence is the sequence of SEQ ID NO:403.

[0397] In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the N-terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein. For example, in some embodiments, CXCR6 or a functional fragment thereof comprises an N- terminal extracellular domain sequence having at least about 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, 28,

39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,

115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the N-terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the N-terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:403. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:403.

[0398] In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the N-terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein. For example, in some embodiments, CXCR6 or a functional fragment thereof comprises an N- terminal extracellular domain sequence having at least about 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, or 30 amino acids deleted from the N-terminal or C-terminal end of the sequence of the N-terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having about 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, or 30 amino acids deleted from the N-terminal or C-terminal end of the sequence of the N-terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having at least about 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, or 30 amino acids deleted from the N- terminal or C-terminal end of the sequence of SEQ ID NO:403. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids deleted from the N- terminal or C-terminal end of the sequence of SEQ ID NO:403. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having at least about 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, or 30 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of the N-terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having about 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, or 30 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of the N- terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having at least about 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, or 30 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:403. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having about 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, or 30 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:403.

[0399] In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:404 or SEQ ID NO:405. In some embodiments, CXCR6 or a functional fragment thereof comprises an N- terminal extracellular region comprising the sequence of SEQ ID NO:404 or SEQ ID NO:405. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:404 or SEQ ID NO:405. In some embodiments, CXCR6 or a functional fragment thereof comprises an N- terminal extracellular region of which sequence is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to SEQ ID NO:404 or SEQ ID NO:405. In some embodiments, CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region of which sequence is the sequence of SEQ ID NO:404 or SEQ ID NO:405.

[0400] Human CXCR6 N-terminal extracellular sequence (amino acid residues 1-32) MAEHDYHEDYGFSSFNDSSQEEHQDFLQFSKV (SEQ ID NO:403)

[0401] Human CXCR6 natural variant E3K N-terminal extracellular sequence (amino acid residues 1-32)

MAKHDYHEDYGFSSFNDSSQEEHQDFLQFSKV (SEQ ID NO:404)

[0402] Human CXCR6 natural variant D25 A N-terminal extracellular sequence (amino acid residues 1-32)

MAEHDYHEDYGFSSFNDSSQEEHQAFLQFSKV (SEQ ID NO:405)

[0403] Human CXCR6 extracellular sequence 2 (amino acid residues 90-103) AGIHEWVFGQVMCK (SEQ ID NO:406)

[0404] Human CXCR6 extracellular sequence 3 (amino acid residues 165-187) PQIIYGNVFNLDKLICGYHDEAI (SEQ ID NO:407)

[0405] Human CXCR6 extracellular sequence 4 (amino acid residues 260-275) YYAMTSFHYTIMVTEA (SEQ ID NO:408)

[0406] In some embodiments, CXCR6 or a functional fragment thereof comprises one or more extracellular domains selected from SEQ ID NO:403-SEQ ID NO:405. In some embodiments, CXCR6 or a functional fragment thereof further comprises one or more extracellular domains selected from SEQ ID NO:406-SEQ ID NO:408. [0407] In some embodiments, CXCR6 or a functional fragment thereof comprises a CXCL16- binding domain.

Transmembrane Domain

[0408] In some embodiments, CXCR6 or a functional fragment thereof is associated with the cell membrane when expressed in a T cell. In some embodiments, CXCR6 or a functional fragment thereof is a membrane-bound protein.

[0409] In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at least one transmembrane domain. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at least two transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at least three transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at least four transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at least five transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at least six transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at least seven transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at most one transmembrane domain. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at most two transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at most three transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at most four transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at most five transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at most six transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at most seven transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising one transmembrane domain. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising two transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising three transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising four transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising five transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising six transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising seven transmembrane domains.

[0410] In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:428. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising the sequence of SEQ ID NO:428. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region of which sequence is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to SEQ ID NO:428. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region of which sequence is the sequence of SEQ ID NO:428.

[0411] In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein. For example, in some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:428. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the sequence of SEQ ID NO:428.

[0412] In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having a deletion of amino acid residue(s) from the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein. For example, in some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids deleted from the N-terminal or C-terminal end of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids deleted from the N-terminal or C-terminal end of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:428. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:428. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids independently deleted from both N- terminal and C-terminal ends of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:428. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids independently deleted from both N-terminal and C- terminal ends of the sequence of SEQ ID NO:428.

[0413] CXCR6 fragment comprising seven transmembrane domains (amino acid residues 33- 293):

FLPCMYLVVFVCGLVGNSLVLVISIFYHKLQSLTDVFLVNLPLADLVFVCTLPFWAYAG IHEWVFGQVMCKSLLGIYTINFYTSMLILTCITVDRFIVVVKATKAYNQQAKRMTWGK VTSLLIWVISLLVSLPQIIYGNVFNLDKLICGYHDEAISTVVLATQMTLGFFLPLLTMIVC YSVIIKTLLHAGGFQKHRSLKIIFLVMAVFLLTQMPFNLMKFIRSTHWEYYAMTSFHYTI MVTEAIAYLRACLNPVLYAFVSL (SEQ ID NO:428)

[0414] CXCR6 transmembrane domain 1 (amino acid residues 33-59): FLPCMYLVVFVCGLVGNSLVLVISIFY (SEQ ID NO:409)

[0415] CXCR6 transmembrane domain 2 (amino acid residues 69-89): FLVNLPLADLVFVCTLPFWAY (SEQ ID NO:410)

[0416] CXCR6 transmembrane domain 3 (amino acid residues 104-125): SLLGIYTINFYTSMLILTCITV (SEQ ID NO:411)

[0417] CXCR6 transmembrane domain 4 (amino acid residues 144-164): RMTWGKVTSLLIWVISLLVSL (SEQ ID NO:412)

[0418] CXCR6 transmembrane domain 5 (amino acid residues 188-215): STWLATQMTLGFFLPLLTMIVCYSVII (SEQ ID NO:413)

[0419] CXCR6 transmembrane domain 6 (amino acid residues 232-259): IIFLVMAVFLLTQMPFNLMKFIRSTHWE (SEQ ID NOAM)

[0420] CXCR6 transmembrane domain 7 (amino acid residues 276-293): IAYLRACLNPVLYAFVSL (SEQ ID NO:415)

[0421] In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to one or more transmembrane domains selected from SEQ ID NO:409-SEQ ID NO:415. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising one or more transmembrane domains selected from SEQ ID NO:409- SEQ ID NO:415.

[0422] In some embodiments, CXCR6 or a functional fragment thereof further comprises (i) one, two, or three cytoplasmic domains, (ii) one, two, or three extracellular domains of CXCR6, or (iii) a combination thereof.

[0423] In some embodiments, CXCR6 or a functional fragment thereof further comprises (i) a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:406, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:407, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:408, or a combination thereof;

(ii) a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:416, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:417, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:418, or a combination thereof; or (iii) any combination thereof. In some embodiments, CXCR6 or a functional fragment thereof further comprises (i) the sequence of SEQ ID NO:406, the sequence of SEQ ID NO:407, the sequence of SEQ ID NO:408, or a combination thereof; (ii) the sequence of SEQ ID NO:416, the sequence of SEQ ID NO:417, the sequence of SEQ ID NO:418, or any combination thereof; or (iii) a combination thereof.

[0424] In some embodiments, CXCR6 or a functional fragment thereof further comprises (i) a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:406, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:407, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:408, or a combination thereof; and (ii) a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:416, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:417, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:418, or a combination thereof. In some embodiments, CXCR6 or a functional fragment thereof further comprises (i) the sequence of SEQ ID NO4:06, the sequence of SEQ ID NO:407, the sequence of SEQ ID NO:408, or a combination thereof; and (ii) the sequence of SEQ ID NO:416, the sequence of SEQ ID NO:417, the sequence of SEQ ID NO:418, or any combination thereof.

Cytoplasmic Domain

[0425] In some embodiments, CXCR6 or a functional fragment thereof comprises at least one cytoplasmic domain. In some embodiments, CXCR6 or a functional fragment thereof comprises at least two cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at least three cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at least four cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at most one cytoplasmic domain. In some embodiments, CXCR6 or a functional fragment thereof comprises at most two cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at most three cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at most four cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises one cytoplasmic domain. In some embodiments, CXCR6 or a functional fragment thereof comprises two cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises three cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises four cytoplasmic domains. [0426] In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:419. In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain comprising the sequence of SEQ ID NO:419. In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain of which sequence is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to SEQ ID NO:419. In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain of which sequence is the sequence of SEQ ID NO:419.

[0427] In some embodiments, the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:419. In some embodiments, the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof comprises the sequence of SEQ ID NO:419. In some embodiments, the sequence of the C- terminal cytoplasmic domain of CXCR6 or a functional fragment thereof is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to SEQ ID NO:419. In some embodiments, , the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof is the sequence of SEQ ID NO:419.

[0428] In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein. For example, in some embodiments, CXCR6 or a functional fragment thereof comprises a C- terminal cytoplasmic domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60,

65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,

170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:419. In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:419.

[0429] In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein. For example, in some embodiments, CXCR6 or a functional fragment thereof comprises a C- terminal cytoplasmic domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, or 45 amino acids deleted from the N-terminal or C-terminal end of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having about 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, 28, 39, 40,

41, 42, 43, 44, or 45 amino acids deleted from the N-terminal or C-terminal end of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having at least about 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, 28, 39, 40, 41,

42, 43, 44, or 45 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:419. In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, or 45 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:419. In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, or 45 amino acids independently deleted from both N-terminal and C- terminal ends of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, or 45 amino acids independently deleted from both N- terminal and C-terminal ends of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein. In some embodiments, CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, or 45 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:419. In some embodiments, CXCR6 or a functional fragment thereof comprises a C- terminal cytoplasmic domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, or 45 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:419. [0430] CXCR6 cytoplasmic domain 1 (amino acid residues 60-68):

HKLQSLTDV (SEQ ID NO:416)

[0431] CXCR6 cytoplasmic domain 2 (amino acid residues 126-143): DRFIVVVKATKAYNQQAK (SEQ ID NO:417)

[0432] CXCR6 cytoplasmic domain 3 (amino acid residues 216-231): KTLLHAGGFQKHRSLK (SEQ ID NO:418)

[0433] CXCR6 C-terminal cytoplasmic domain (amino acid residues 294-342): KFRKNFWKLVKDIGCLPYLGVSHQWKSSEDNSKTFSASHNVEATSMFQL (SEQ ID NO:419)

[0434] In some embodiments, CXCR6 or a functional fragment thereof comprises a cytoplasmic domain comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to one or more cytoplasmic domain selected from SEQ ID NO:416 SEQ ID NO:419. In some embodiments, CXCR6 or a functional fragment thereof comprises a cytoplasmic domain comprising one or more cytoplasmic domain selected from SEQ ID NO:416 SEQ ID NO:419.

Examples of CXCR6 construct

[0435] In some embodiments, the recombinant nucleic acid comprising CXCR6 or a functional fragment thereof as described herein comprises a sequence encoding an amino acid sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:420, SEQ ID NO:424, SEQ ID NO:426, or SEQ ID NO:435. In some embodiments, the recombinant nucleic acid comprising CXCR6 or a functional fragment thereof as described herein comprises a sequence encoding the sequence of SEQ ID NO:420, SEQ ID NO:424, SEQ ID NO:426, or SEQ ID NO:435. In some embodiments, the recombinant nucleic acid comprising CXCR6 or a functional fragment thereof as described herein comprises the sequence of SEQ ID NO:426. In some embodiments, the sequence of the recombinant nucleic acid comprising CXCR6 or a functional fragment thereof as described herein is a sequence encoding an amino acid sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:420, SEQ ID NO:424, SEQ ID NO:426, or SEQ ID NO:435. In some embodiments, the sequence of the recombinant nucleic acid comprising CXCR6 or a functional fragment thereof as described herein is a sequence encoding the sequence of SEQ ID NO:420, SEQ ID NO:424, SEQ ID NO:426, or SEQ ID NO:435. [0436] In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein further comprises a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof. In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein further comprises a sequence encoding the amino acid sequence of SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof. In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein further encodes an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof. In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein further encodes the amino acid sequence of SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof.

T-cell Receptor (TCR) Fusion Proteins (TFPs)

[0437] 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 antibody or antibody fragment can comprise an antigen binding domain 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-CD79b 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 B AFF 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. The present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to a tumor associated antigen (a 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 DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD 19, e.g., human CD 19, 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, e.g., DNA, 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 present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to MUC16, e.g., human MUC16, 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 DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD20, e.g., human CD20, 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 DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD70, e.g., human CD70, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof. The present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD79B, e.g., human CD79B, 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 DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to HER2, e.g., human HER2, 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 DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to PSMA, e.g., human PSMA, 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 DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to BCMA, e.g., human BCMA, 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 DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to R0R1, e.g., human R0R1, 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 DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD22, e.g., human CD22, 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 DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to GPC3, e.g., human GPC3, 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 DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to Nectin-4, e.g., human Nectin-4, 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 DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to Trop-2, e.g., human Trop-2, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof. The TFPs provided herein are able to associate with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR subunits in order to form a functional TCR complex.

[0438] 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).

[0439] 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.

[0440] 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 (VL) and a variable domain (VHH) of a camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen binding domain, such as a recombinant fibronectin domain, 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 embodiments, 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.

[0441] Thus, in one aspect, the antigen-binding domain comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the murine, humanized or human anti-TAA binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a murine, humanized or human anti-TAA binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a murine, humanized or human anti-TAA 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-TAA binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a 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 embodiments, the linker sequence comprises a long linker (LL) sequence. In some embodiments, the long linker sequence comprises (G4S)_n, wherein n=2 to 4. In some embodiments, the linker sequence comprises a short linker (SL) sequence. In some embodiments, the short linker sequence comprises (G4S)n, wherein n=l to 3.

[0442] In some embodiments, the antigen binding domain is an antibody or a fragment thereof. In some embodiments, the antigen binding domain is a camelid antibody or a binding fragment thereof. In some embodiments, the antigen binding domain is a murine antibody or a binding fragment thereof. In some embodiments, the antigen binding domain is a human or humanized antibody or a binding fragment thereof. In some embodiments, the antigen binding domain is a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain. In some embodiments, the sdAb is a VHH.

[0443] In some embodiments, the antigen binding domain is selected from the 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, anti-MUC16 binding domain, an anti-Nectin-4 binding domain, an anti-GPC3 binding domain, and an anti-TROP-2 binding domain.

[0444] 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 CD 19. In one aspect, the antigen binding domain has the same or a similar binding specificity to human CD19 as the FMC63 scFv described in Nicholson et al., Mol. Immun. 34 (16-17): 1157-1165 (1997). 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 CD 19 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.

[0445] In some embodiments, the anti-CD19 antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences listed in Table 5. In some embodiments, the anti-CD19 antibody or fragment thereof as described herein comprises any one of the CDR sequences listed in Table 5. In some embodiments, the anti-CD19 antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences of the anti-CD19 antibody or fragment thereof sequences listed in Table 5. In some embodiments, the anti-CD19 antibody or fragment thereof as described herein comprises a CDR sequence of any one of the CDR sequences of the anti-CD19 antibody or fragment thereof sequences listed in Table 5. In some embodiments, the anti-CD19 antibody or fragment thereof as described herein comprises an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the anti-CD19 antibody or fragment thereof sequences listed in Table 5. In some embodiments, the anti-CD19 antibody or fragment thereof as described herein comprises any one of the anti-CD19 antibody or fragment thereof sequences listed in Table 5.

[0446] In some embodiments, the antigen-binding domain comprises an anti-CD19 humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment having a light chain CDR1 of SEQ ID NO:73, a CDR2 of SEQ ID NO:75, and a CDR3 of SEQ ID NO: 77 and a heavy chain CDR1 of SEQ ID NO: 79, a CDR2 of SEQ ID NO: 81, and a CDR3 of SEQ ID NO:83. In some embodiments, the anti-CD19 antibody is a murine scFv. In some embodiments, the anti-CD-19 antibody comprises a VL of SEQ ID NO:85 and a VH of SEQ ID NO:87.

[0447] In one embodiment, the antibody has the antigen binding domain of an anti-mesothelin antibody. Exemplary antibodies that bind mesothelin include, but are not limited to, amatuximab and those described in W02006099141, WO2006124641, W02009120769, WO2010111282, W02014004549, WO2014031476, W02014052064, WO2017032293, and WO2017052241, the contents of each of which are incorporated by reference herein in their entirety.

[0448] In some embodiments, the anti-mesothelin antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences listed in Table 5. In some embodiments, the anti-mesothelin antibody or fragment thereof as described herein comprises any one of the CDR sequences listed in Table 5. In some embodiments, the anti-mesothelin antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences of the anti-mesothelin antibody or fragment thereof sequences listed in Table 5. In some embodiments, the anti-mesothelin antibody or fragment thereof as described herein comprises a CDR sequence of any one of the CDR sequences of the anti-mesothelin antibody or fragment thereof sequences listed in Table 5. In some embodiments, the anti-mesothelin antibody or fragment thereof as described herein comprises an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the anti-mesothelin antibody or fragment thereof sequences listed in Table 5. In some embodiments, the anti-mesothelin antibody or fragment thereof as described herein comprises any one of the anti-mesothelin antibody or fragment thereof sequences listed in Table 5.

[0449] In some embodiments, the antigen-binding domain comprises an anti-mesothelin 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.

[0450] In one embodiment, the antibody has the antigen binding domain of an anti-CD70 antibody. Exemplary antibodies that bind CD70 include, but are not limited to, cusatuzumab, MDX-1411, vorsetuzumab and those described in WO2014158821 and WO2018152181, the contents of each of which are incorporated by reference herein in their entirety.

[0451] In some embodiments, the anti-CD70 antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences listed in Table 5. In some embodiments, the anti-CD70 antibody or fragment thereof as described herein comprises any one of the CDR sequences listed in Table 5. In some embodiments, the anti-CD70 antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences of the anti-CD70 antibody or fragment thereof sequences listed in Table 5. In some embodiments, the anti-CD70 antibody or fragment thereof as described herein comprises a CDR sequence of any one of the CDR sequences of the anti-CD70 antibody or fragment thereof sequences listed in Table 5. In some embodiments, the anti-CD70 antibody or fragment thereof as described herein comprises an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the anti-CD70 antibody or fragment thereof sequences listed in Table 5. In some embodiments, the anti-CD70 antibody or fragment thereof as described herein comprises any one of the anti-CD70 antibody or fragment thereof sequences listed in Table 5.

[0452] In some embodiments, the antigen-binding domain comprises an anti-CD70 humanized or human single domain antibody or an antibody fragment having a CDR1 of SEQ ID NO:88, a

CDR2 of SEQ ID NO: 89, and a CDR3 of SEQ ID NO: 90, or a CDR1 of SEQ ID NO: 92, a

CDR2 of SEQ ID NO: 93, and a CDR3 of SEQ ID NO: 94, or a CDR1 of SEQ ID NO: 96, a

CDR2 of SEQ ID NO: 97, and a CDR3 of SEQ ID NO: 98, or a CDR1 of SEQ ID NO: 100, a

CDR2 of SEQ ID NO: 101, and a CDR3 of SEQ ID NO: 102, or a CDR 1 of SEQ ID NO: 104, a

CDR2 of SEQ ID NO: 105, and a CDR3 of SEQ ID NO: 106, or a CDR1 of SEQ ID NO: 108, a

CDR2 of SEQ ID NO: 109, and a CDR3 of SEQ ID NO: 110, or a CDR1 of SEQ ID NO: 112, a

CDR2 of SEQ ID NO: 113, and a CDR3 of SEQ ID NO: 114, or a CDR1 of SEQ ID NO: 116, a

CDR2 of SEQ ID NO: 117, and a CDR3 of SEQ ID NO: 118, or a CDR1 of SEQ ID NO: 120, a

CDR2 of SEQ ID NO: 121, and a CDR3 of SEQ ID NO: 122.

[0453] In some embodiments, the antigen-binding domain comprises an anti-CD70 single chain Fv (scFv) or an antibody fragment thereof. The anti-CD70 scFv or antibody fragment thereof can comprise a heavy chain complementary determining region 1 (CDRH1) having a sequence of SEQ ID NO:361, a CDRH2 having a sequence of SEQ ID NO: 362, and a CDRH3 having a sequence of SEQ ID NOs:363. The anti-CD70 scFv or antibody fragment thereof can comprise a light chain complementary determining region 1 (CDRL1) having a sequence of SEQ ID NO:365, a CDRL2 having a sequence of SEQ ID NO:366, and a CDRL3 having a sequence of SEQ ID NO:367. The anti-CD70 scFv or antibody fragment thereof can comprise a heavy chain variable (VH) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:364. The anti-CD70 scFv or antibody fragment thereof can comprise a light chain variable (VL) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 368.

[0454] In one embodiment, the antibody has the antigen binding domain of an anti-BCMA antibody. Exemplary antibodies that bind BCMA include, but are not limited to, SEA-BCMA (Seattle Genetics) and those described in W02010104949, WO2011108008, WO2014122143, W02016090327, WO2017143069, WO2017211900, WO2018133877, WO2019066435, WO2019149269. WO2019190969, and W02019195017, the contents of each of which are incorporated by reference herein in their entirety.

[0455] In some embodiments, the anti-BCMA antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences listed in Table 5. In some embodiments, the anti-BCMA antibody or fragment thereof as described herein comprises any one of the CDR sequences listed in Table 5. In some embodiments, the anti-BCMA antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences of the anti-BCMA antibody or fragment thereof sequences listed in Table 5. In some embodiments, the anti-BCMA antibody or fragment thereof as described herein comprises a CDR sequence of any one of the CDR sequences of the anti-BCMA antibody or fragment thereof sequences listed in Table 5. In some embodiments, the anti-BCMA antibody or fragment thereof as described herein comprises an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the anti-BCMA antibody or fragment thereof sequences listed in Table 5. In some embodiments, the anti-BCMA antibody or fragment thereof as described herein comprises any one of the anti-BCMA antibody or fragment thereof sequences listed in Table 5.

[0456] In one embodiment, the antibody has the antigen binding domain of an anti- MUC16 antibody. Exemplary antibodies that bind MUC16 include, but are not limited to, oregovomab, 4H11 (Memorial Sloan Kettering Cancer Center), sofituzumab, and those described in W02018058003, the contents of which is incorporated by reference herein in its entirety.

[0457] In one embodiment, the antibody has the antigen binding domain of an anti- CD79B antibody. Exemplary antibodies that bind CD79B include, but are not limited to, those described in W02017009474 and W02016021621, the contents of each of which are incorporated by reference herein in their entirety. [0458] In one embodiment, the antibody has the antigen binding domain of an anti-HER2 antibody. Exemplary antibodies that bind HER2 include, but are not limited to, trastuzumab, pertuzumab, margetuximab, trastuzumab-pkrb, ertumaxomab, SB3, PF-05280014, CMAB302, trastuzumab-dkst, HD201, GB221, BCD-022, trastuzumab-anns, HLX02, DMB-3111, timigutuzumab, UB-921, IBB 15, RG6194, HLX22, SIBP-01, TX05, and DXL702.

[0459] In one embodiment, the antibody has the antigen binding domain of an anti-PSMA antibody. Exemplary antibodies that bind PSMA include, but are not limited to, MDX1201- A488 and those described in W02001009192, W0200303490, W02007002222, W02009130575, WO2010118522, WO2013185117, WO2013188740, WO2014198223, WO2016145139, W02017121905, W02017180713, WO2017212250, WO2018033749, WO2018129284, WO2018142323, and WO2019191728, the contents of each of which are incorporated by reference herein in their entirety.

[0460] In some embodiments, the antigen-binding domain or fragment thereof as described herein comprises a sequence encoding an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the amino acid sequences listed in Table 5. In some embodiments, the antigen-binding domain or fragment thereof as described herein comprises a sequence encoding any one of the amino acid sequences listed in Table 5. In some embodiments, the antigen-binding domain or fragment thereof as described herein comprises a nucleic acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the nucleic acid sequences listed in Table 5. In some embodiments, the antigen-binding domain or fragment thereof as described herein comprises a nucleic acid sequence encoding any one of the nucleic acid sequences listed in Table 5. In some embodiments, the antigen-binding domain or fragment thereof as described herein comprises a sequences encoding CDR sequences having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences listed in Table 5. In some embodiments, the antigen-binding domain or fragment thereof as described herein comprises a sequence encoding any one of the CDR sequences listed in Table 5. In some embodiments, the antigen-binding domain or fragment thereof as described herein comprises a sequence encoding CDR sequences having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to the CDR sequences of any one of the sequences listed in Table 5. In some embodiments, the antigen-binding domain or fragment thereof as described herein comprises a sequence encoding CDR sequences of any one of the sequences listed in Table 5.

[0461] 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 tumor-associated 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 binding domain is contiguous with and in the same reading frame as a leader sequence.

[0462] 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.

[0463] A humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g, European Patent No. EP 239,400;

International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g, European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805- 814; and Roguska et al., 1994, PNAS, 91 :969-973, each of which is incorporated herein by its entirety by reference), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in its entirety by reference), and techniques disclosed in, e.g., U.S. Patent Application Publication No. US2005/0042664, U.S. Patent Application Publication No. US2005/0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169: 1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein in its entirety by reference. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, for example improve, antigen binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.)

[0464] 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, 332'323-321 (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.

[0465] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151 :2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (see, e.g., Nicholson et al., Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151 :2623 (1993), the contents of which are incorporated herein by reference herein in their entirety). In some embodiments, the framework region, e.g., all four framework regions, of the heavy chain variable region are derived from a VH -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.

[0466] 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.

[0467] A humanized antibody or antibody fragment may retain a similar antigenic specificity as the original antibody, e.g., in the present disclosure, the ability to bind human a tumor associated antigen (TAA). In some embodiments, a humanized antibody or antibody fragment may have improved affinity and/or specificity of binding to, e.g., human CD 19, human BCMA, human MUC16, human mesothelin (MSLN), human CD79B, human HER2, human PSMA, human CD20, human CD70, human Nectin-4, human GPC3, human TROP-2, human PD-1, or another tumor associated antigen.

[0468] 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).

[0469] Also provided herein are methods for obtaining an antibody antigen binding domain specific for a target antigen (e.g, CD 19, BCMA, MUC16, MSLN, CD20, CD70, CD79B, HER2, PSMA, Nectin-4, GPC3, TROP-2, PD-1, or any target antigen described elsewhere herein for targets of fusion moiety binding domains), the method comprising providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a VH 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, CD79B, etc.) and optionally with one or more desired properties.

[0470] In some embodiments, VH domains and scFvs can be prepared according to method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). scFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The linker length can greatly affect how the variable regions of a scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5-10 amino acids) intra-chain folding is prevented. Inter-chain folding is also required to bring the two variable regions together to form a functional epitope binding site. In some embodiments, the linker sequence comprises a linker sequence. In some embodiments, the long linker sequence comprises (G4S)_n, wherein n=2 to 4. In some embodiments, 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.

[0471] 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 embodiments, the linker sequence comprises (GiS)n, wherein n=2 to 4. In some embodiments, the linker sequence comprises (GiS)n, wherein n=l to 3. In some embodiments, the linker sequence comprises GGSGGSGGSGGS (SEQ ID NO: 369).

Stability and Mutations

[0472] The stability of a tumor associated antigen binding domain, e.g., scFv or sdAb molecules (e.g., soluble scFv or sdAb) can be evaluated in reference to the biophysical properties (e.g., thermal stability) of a conventional control scFv or sdAb molecule or a full-length antibody. In one embodiment, the humanized or human scFv or sdAb 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 or sdAb in the described assays.

[0473] The improved thermal stability of the anti-TAA binding domain, e.g., scFv or sdAb 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 or sdAb 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 or sdAb molecules as described herein and scFv or sdAb 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.

[0474] Mutations in antibody sequences (arising through humanization or direct mutagenesis of the soluble scFv or sdAb) alter the stability of the antibody or fragment thereof and improve the overall stability of the antibody and the 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 or sdAb 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.

[0475] 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 or sdAb.

[0476] 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.

[0477] 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.

[0478] 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). [0479] 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.

[0480] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. 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.

[0481] In one aspect, the present disclosure contemplates modifications of the starting antibody or fragment (e.g., scFv or sdAb) amino acid sequence that generate functionally equivalent molecules. For example, the VH or VL of a binding domain, e.g., scFv or sdAb, comprised in the TFP can be modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting VH or VL framework region of the antibody fragment as described herein, e.g., scFv or sdAb. 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

[0482] 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 embodiments, 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.

[0483] In some embodiments, the TCR extracellular domain comprises an extracellular domain or portion thereof of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain. In some embodiments, the TCR extracellular domain comprises the extracellular portion of a constant (an IgC) domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain.

[0484] 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, 150 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%, 99.5%, 99.7%, 99.9% 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. [0485] 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, 150 or more consecutive amino acid residues of the extracellular portion of a constant (an IgC) domain of TCR alpha, a TCR beta, a

TCR delta, or a TCR gamma. In some embodiments, the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to a sequence encoding the extracellular portion of a constant (an IgC) domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma. In some embodiments, the extracellular domain comprises a sequence encoding the extracellular portion of a constant (an IgC) domain of TCR alpha, TCR beta, TCR delta, or TCR gamma having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C- terminus.

[0486] 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%, 99.5%, 99.7%, 99.9% 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.

[0487] The extracellular domain can comprise a full-length extracellular portion of the 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 extracellular portion of the 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 extracellular portion of the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.

[0488] 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.

[0489] 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

[0490] 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 embodiments, 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.

[0491] 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 embodiments, 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, CD 16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0492] 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%, 99.5%, 99.7%, 99.9% 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.

[0493] In some embodiments, 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

[0494] Optionally, a short oligo- or polypeptide linker, between 2 and 10 amino acids in length may form the linkage between the binding element and the TCR extracellular domain of the TFP. A glycine-serine doublet provides a particularly suitable linker. In some cases, the linker may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more in length. For example, in one aspect, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:429) 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 (SEQ ID NO:430). 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 (SEQ ID NO:572).

Cytoplasmic Domain

[0495] 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,” as used herein, 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.

[0496] 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%, 99.5%, 99.7%, 99.9% 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. [0497] 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%, 99.5%, 99.7%, 99.9% 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.

[0498] It is known that signals generated through the TCR alone are insufficient for full activation of naive T cells and that a secondary and/or costimulatory signal is required. Thus, naive T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic domain, e.g., a costimulatory domain).

[0499] 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 tyrosinebased activation motifs (ITAMs).

[0500] 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 IT AM domain, e.g., a mutated IT AM 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 IT AM motifs.

[0501] 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).

[0502] The intracellular signaling sequences within the cytoplasmic portion of the TFP of the present disclosure may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences.

[0503] 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. [0504] 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.

[0505] 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.

[0506] In one aspect, the TFP-expressing cell that co-expresses CXCR6 or a functional fragment thereof as 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 or a different target. 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.

[0507] TFP constructs can be generated as previously described. An anti-MSLN or CD 19 binder can be linked to a CD3 or TCR DNA fragment by either a DNA sequence encoding a short linker (SL): AAAGGGGSGGGGSGGGGSLE (SEQ ID NO:387) or a long linker (LL): AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE (SEQ ID NO:388) into pLRPO or p510 vector. In some embodiments, the TFP used is TC-110 (e.g., an anti-CD19 FMC63 scFv antibody linked to CD3 epsilon) having the sequence of SEQ ID NO: 196.

[0508] In some embodiments, the TFP used comprises an anti-MSLN antibody and CD3. In some embodiments, the TFP used comprises an anti-MSLN antibody linked to CD3. In some embodiments, the TFP used comprises an anti-MSLN antibody operatively linked to CD3. In some embodiments, the TFP used comprises an anti-MSLN MHle VHH antibody and CD3. In some embodiments, the TFP used comprises an anti-MSLN MHle VHH antibody linked to CD3. In some embodiments, the TFP used comprises an anti-MSLN MHle VHH antibody operatively linked to CD3. In some embodiments, the TFP used comprises an anti-MSLN MHle VHH antibody and CD3 epsilon. In some embodiments, the TFP used comprises an anti-MSLN MHle VHH antibody linked to CD3 epsilon. In some embodiments, the TFP used comprises an anti-MSLN MHle VHH antibody operatively linked to CD3 epsilon.

[0509] In some embodiments, the TFP used is TC-210 comprising an anti-MSLN antibody and CD3. In some embodiments, the TFP used is TC-210 comprising an anti-MSLN antibody linked to CD3. In some embodiments, the TFP used is TC-210 comprising an anti-MSLN antibody operatively linked to CD3. In some embodiments, the TFP used is TC-210 comprising an anti- MSLN MHle VHH antibody and CD3. In some embodiments, the TFP used is TC-210 comprising an anti-MSLN MHle VHH antibody linked to CD3. In some embodiments, the TFP used is TC-210 comprising an anti-MSLN MHle VHH antibody operatively linked to CD3. In some embodiments, the TFP used is TC-210 comprising an anti-MSLN MHle VHH antibody and CD3 epsilon. In some embodiments, the TFP used is TC-210 comprising an anti-MSLN MHle VHH antibody linked to CD3 epsilon. In some embodiments, the TFP used is TC-210 comprising an anti-MSLN MHle VHH antibody operatively linked to CD3 epsilon.

[0510] In some embodiment, the TFP used comprises a GM-CSFRa Signal Peptide, an anti- MSLN MHle VHH antibody, a A3(G4S)3LE Linker, and CD3 epsilon. In some embodiment, the TFP used comprises a GM-CSFRa Signal Peptide operatively linked to an anti-MSLN MHle VHH antibody operatively linked to a A3(G4S)3LE Linker operatively linked to CD3 epsilon. In some embodiment, the TFP used comprises 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 SEQ ID NO:421, 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 SEQ ID NO:422, 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 SEQ ID NO:387, and 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 SEQ ID NO:423. In some embodiment, the TFP used comprises 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 SEQ ID NO:421 operatively linked to 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 SEQ ID NO:422 operatively linked to 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 SEQ ID NO: 387 operatively linked to 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 SEQ ID NO:423. In some embodiment, the TFP used comprises the sequence of SEQ ID NO:421, the sequence of SEQ ID NO:422, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:423. In some embodiment, the TFP used comprises the sequence of SEQ ID NO:421 operatively linked to the sequence of SEQ ID NO:422 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:423.

[0511] In some embodiments, the TFP used comprises 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: 195. In some embodiments, the TFP used comprises the sequence of SEQ ID NO: 195. In some embodiments, the TFP used is TC-210 comprising 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: 195. In some embodiments, the TFP used is TC-210 comprising the sequence of SEQ ID NO: 195. In some embodiments, the TFP used is TC-210 (e.g., an anti-MSLN MHle VHH antibody linked to CD3 epsilon) having the sequence of SEQ ID NO: 195.

[0512] Anti-MSLN-CD3 epsilon (SEQ ID NO: 195)

MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGGDWSANFMYW YRQAPGKQRELVARISGRGVWYVESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY CAVASYWGQGTLVTVSSAAAGGGGSGGGGSGGGGSLEDGNEEMGGITQTPYKVSISG TTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRG SKPEDANFYI.,YI,RARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAK PVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI [0513] Anti-CD19-CD3 epsilon (SEQ ID NO: 196) MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQ KPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFG GGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVS WTRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQWLKMNSLQTDDTAIYY CAKHYYYGGSYAMDYWGQGTSVTVSSAAAGGGGSGGGGSGGGGSLEDGNEEMGGIT QTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQS GYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYW SKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

Recombinant Nucleic Acid Molecules

[0514] Disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) as described herein and a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein.

Recombinant Nucleic Acid Encoding a TFP and a TCR Constant Domain

[0515] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP) and a sequence encoding CXCR6 or a fragment thereof, wherein the recombinant nucleic acid further expresses a TCR constant domain . 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.

[0516] 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.

[0517] 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.

[0518] 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:21, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO:207, SEQ ID NO:209, or 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: 16, SEQ ID NO:21, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO:207, SEQ ID NO:209, or 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: 16, SEQ ID NO:21, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO:207, SEQ ID NO:209, or SEQ ID NO:243. The constant domain can comprise a sequence or fragment thereof of SEQ ID NO: 16, SEQ ID NO:21, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO:207, SEQ ID NO:209, or 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: 16, SEQ ID NO:21, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO:207, SEQ ID NO:209, or 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: 16, SEQ ID NO:21, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO:207, SEQ ID NO:209, or 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: 16, SEQ ID NO:21, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO:207, SEQ ID NO:209, or SEQ ID NO:243.

[0519] 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.

[0520] 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.

[0521] In some embodiments, 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. [0522] 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.

[0523] 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.

[0524] 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.

[0525] 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.

[0526] 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.

[0527] In some embodiments, 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. [0528] In some embodiments, 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. [0529] In some embodiments, 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.

[0530] In some embodiments, the TCR constant domain incorporates into a functional TCR complex when expressed in a T cell. In some embodiments, 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 embodiments, the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained within a same nucleic acid molecule. In some embodiments, 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.

[0531] T2A cleavage site: EGRGSLLTCGDVEENPGP (SEQ ID NO:23).

[0532] 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.

[0533] In some embodiments, 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 embodiments, the linker sequence comprises (G4S)_n, wherein n=l to 4.

[0534] 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.

[0535] In some embodiments, 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: 142, SEQ ID NO: 143, SEQ ID NO: 146, or 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 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: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO:209, or 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.

[0536] 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: 142, SEQ ID NO: 143, Or SEQ ID NO: 146, 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: 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.

[0537] 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 CD3 epsilon gene sequence.

[0538] 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.

[0539] In some embodiments, 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 embodiments, (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. [0540] 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 embodiments, the murine, human or humanized antibody 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.

[0541] 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-CD79b binding domain, an anti-PMS A binding domain, an anti-MUC16 binding domain, an anti-CD22 binding domain, an anti-PD-Ll binding domain, an anti 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.

[0542] In some embodiments, the nucleic acid is selected from the group consisting of a DNA and an RNA. In some embodiments, the nucleic acid is an mRNA. In some embodiments, 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 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’-O-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.

[0543] In some embodiments, the recombinant nucleic acid further comprises a leader sequence. In some embodiments, the recombinant nucleic acid further comprises a promoter sequence. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some embodiments, the recombinant nucleic acid further comprises a 3’UTR sequence. In some embodiments, the nucleic acid 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.

[0544] The present disclosure, in some cases, provides a recombinant nucleic acid comprising: a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked, and a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain. In some embodiment, a modified T cell further comprises a functional disruption of an endogenous TCR.

[0545] The present disclosure, in some cases, provides a recombinant nucleic acid comprising (a) a 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, (2) a transmembrane domain, and (3) an intracellular domain of TCR alpha, TCR beta, TCR gamma, or TCR delta or an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta, and; and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked, and a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain. In some embodiment, a modified T cell further comprises a functional disruption of an endogenous TCR.

[0546] 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’-0-M0E), 2’-O- aminopropyl, 2’-deoxy, T-deoxy -2 ’-fluoro, 2’-O-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.

TGFBrl Switch Polypeptides

[0547] Provided herein are recombinant nucleic acids comprising: a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked, and a second nucleic acid sequence encoding C-X-C chemokine receptor type 6 (CXCR6) or a functional fragment thereof. In some embodiments, the recombinant nucleic acids as described herein further comprises a third nucleic acid sequence encoding a TGFBr2 switch polypeptide comprising a transforming growth factor beta receptor II (TGFBr2) extracellular domain or a functional fragment thereof. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a linker. In some embodiments, the linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A cleavage site. In some embodiments, the cleavage site can be a self-cleaving peptide such as a T2A, P2A, E2A or F2A cleavage site. In some embodiments, the 2A cleavage site is a T2A cleavage site or a P2A cleavage site. In some embodiments, the third nucleic acid sequence are operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof. In some embodiments, the third nucleic acid sequence are operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof by a linker. In some embodiments, the third nucleic acid sequence are operatively linked to the first nucleic acid sequence, and/or the second nucleic acid sequence, independently. In some embodiments, the third nucleic acid sequence are operatively linked to the first nucleic acid sequence, and/or the second nucleic acid sequence, independently, by a linker. In some embodiments, the linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A cleavage site. In some embodiments, the 2A cleavage site is a T2A cleavage site or a P2A cleavage site. In some embodiments, the third nucleic acid sequence and the first nucleic acid sequence, the third nucleic acid sequence and the second nucleic acid sequence, or the third nucleic acid sequence, the first nucleic acid sequence, and the second nucleic acid sequence are present on different nucleic acid molecules.

[0548] In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs:283, 284, 285, and 286. In some embodiments, the TGFBr2 switch polypeptide comprises any one sequence selected from SEQ ID NOs:283, 284, 285, and 286. In some embodiments, the sequence of the TGFBr2 switch polypeptide is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs:283, 284, 285, and 286. In some embodiments, the sequence of the TGFBr2 switch polypeptide is any one sequence selected from SEQ ID NOs:283, 284, 285, and 286.

[0549] In some embodiments, the TGFBr2 switch polypeptide comprises an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of a TGFBr2 switch polypeptide as described herein. For example, in some embodiments, the TGFBr2 switch polypeptide comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of a TGFBr2 switch polypeptide as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of a TGFBr2 switch polypeptide as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of any one sequence selected from SEQ ID NOs:283, 284, 285, and 286. In some embodiments, the TGFBr2 switch polypeptide comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of any one sequence selected from SEQ ID NOs:283, 284, 285, and 286.

[0550] In some embodiments, the TGFBr2 switch polypeptide comprises amino acid residue deletions from the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of a TGFBr2 switch polypeptide as described herein. For example, in some embodiments, the TGFBr2 switch polypeptide comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C-terminal end of a sequence of a TGFBr2 switch polypeptide as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C-terminal end of a sequence of a TGFBr2 switch polypeptide as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C- terminal end of any one sequence selected from SEQ ID NOs:283, 284, 285, and 286. In some embodiments, the TGFBr2 switch polypeptide comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C-terminal end of any one sequence selected from SEQ ID NOs:283, 284, 285, and 286. In some embodiments, the TGFBr2 switch polypeptide comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of a sequence of a TGFBr2 switch polypeptide as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of a sequence of a TGFBr2 switch polypeptide as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of any one sequence selected from SEQ ID NOs:283, 284, 285, and 286. In some embodiments, the TGFBr2 switch polypeptide comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of any one sequence selected from SEQ ID NOs:283, 284, 285, and 286.

[0551] In some embodiments, the TGFBr2 extracellular domain comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437. In some embodiments, the TGFBr2 extracellular domain comprises the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437. In some embodiments, the sequence of the TGFBr2 extracellular domain is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437. In some embodiments, the sequence of the TGFBr2 extracellular domain is the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437.

[0552] In some embodiments, the TGFBr2 switch polypeptide comprises an extracellular domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the TGFBr2 extracellular domain as described herein. For example, in some embodiments, the TGFBr2 switch polypeptide comprises an extracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the sequence of the TGFBr2 extracellular domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an extracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the TGFBr2 extracellular domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an extracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437. In some embodiments, the TGFBr2 switch polypeptide comprises an extracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437.

[0553] In some embodiments, the TGFBr2 switch polypeptide comprises an extracellular domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of the TGFBr2 extracellular domain as described herein. For example, in some embodiments, the TGFBr2 switch polypeptide comprises an extracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids deleted from the N-terminal or C- terminal end of the sequence of the TGFBr2 extracellular domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an extracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids deleted from the N-terminal or C-terminal end of the sequence of the TGFBr2 extracellular domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an extracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437. In some embodiments, the TGFBr2 switch polypeptide comprises an extracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437. In some embodiments, in some embodiments, the TGFBr2 switch polypeptide comprises an extracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of the TGFBr2 extracellular domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an extracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of the TGFBr2 extracellular domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an extracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437. In some embodiments, the TGFBr2 switch polypeptide comprises an extracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437.

[0554] TGFBr2 extracellular domain (TGFBR2 ecto): TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQE VCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPDLLLVIFQ (SEQ ID NO:271)

[0555] TGFBr2 extracellular domain with a signal peptide: MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAAS PKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ (SEQ ID NO:432) [0556] TGFBr2 signal peptide: MGRGLLRGLWPLHIVLWTRIAS (SEQ ID NO:431)

Switch Intracellular Domain

[0557] In some embodiments, the TGFBr2 switch polypeptide further comprises a switch intracellular domain. In some embodiments, the TGFBr2 extracellular domain or functional fragment thereof is operably linked to the switch intracellular domain. In some embodiments, the switch intracellular domain comprises an intracellular domain of a costimulatory polypeptide. In some embodiments, the costimulatory polypeptide is selected from the group consisting of CD28, 4-lBB(CD137), IL-15Ra, IL12R, IL18R, IL21R, 0X40, CD2, CD27, CD5, ICAM-1, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, CD226, FcyRI, FcyRII, and FcyRIII. In some embodiments, the costimulatory polypeptide is CD28. In some embodiments, the costimulatory polypeptide is 4- 1BB. In some embodiments, the costimulatory polypeptide is IL-15Ra.

[0558] In some embodiments, the switch intracellular domain comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to or SEQ ID NO:273 or SEQ ID NO:277. In some embodiments, the switch intracellular domain comprises the sequence of SEQ ID NO:273 or SEQ ID NO:277. In some embodiments, the sequence of the switch intracellular domain is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:273 or SEQ ID NO:277. In some embodiments, the sequence of the switch intracellular domain is the sequence of SEQ ID NO:273 or SEQ ID NO:277. [0559] In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the intracellular domain sequence as described herein. For example, in some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the intracellular domain sequence as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the intracellular domain sequence as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N- terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:273 or SEQ ID NO:277. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C- terminal ends of the sequence of SEQ ID NO:273 or SEQ ID NO:277.

[0560] In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the intracellular domain sequence as described herein. For example, in some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 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, or 35 amino acids deleted from the N-terminal or C-terminal end of the intracellular domain sequence as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 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, or 35 amino acids deleted from the N-terminal or C-terminal end of the intracellular domain sequence as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 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, or 35 amino acids deleted from the N-terminal or C- terminal end of the sequence of SEQ ID NO:273 or SEQ ID NO:277. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 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, or 35 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:273 or SEQ ID NO:277. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 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, or 35 amino acids independently deleted from both N-terminal and C-terminal ends of the intracellular domain sequence as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 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, or 35 amino acids independently deleted from both N-terminal and C-terminal ends of the intracellular domain sequence as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 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, or 35 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:273 or SEQ ID NO:277. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 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, or 35 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:273 or SEQ ID NO:277.

[0561] In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one selected from SEQ ID NOs:287, 277, 288, 289, 273, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 313, 314, 315, 316, and a combination thereof. In some embodiments, the TGFBr2 switch polypeptide comprises any one sequence selected from the group consisting of SEQ ID NOs:287, 277, 288, 289, 273, 290, 291, 292, 293, 294, 295, 296,

297, 298, 299, 313, 314, 315, 316, and a combination thereof. In some embodiments, the sequence of the switch intracellular domain is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one selected from SEQ ID NOs:287, 277, 288, 289, 273, 290, 291, 292, 293, 294, 295, 296, 297,

298, 299, 313, 314, 315, 316, and a combination thereof. In some embodiments, the sequence of the switch intracellular domain is any one sequence selected from the group consisting of SEQ ID NOs:287, 277, 288, 289, 273, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 313, 314, 315, 316, and a combination thereof.

[0562] PD -1 Intracellular domain CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATI VFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO:287) [0563] 4-1BB Intracellular domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:277) [0564] ICOS Intracellular domain CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO:288) [0565] CTLA4 Intracellular domain AVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO:289) [0566] CD28 Intracellular domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:273) [0567] CD200R Intracellular domain KVNGCRKYKLNKTESTPVVEEDEMQPYASYTEKNNPLYDTTNKVKASEALQSEVDTD LHTL (SEQ ID NO:290)

[0568] BTLA Intracellular domain RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRM QEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS (SEQ ID NO:291)

[0569] TIM-3 Intracellular domain FKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYV SSRQQPSQPLGCRFAMP (SEQ ID NO:292)

[0570] TIGIT Intracellular domain LTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCA ELHDYFNVLSYRSLGNCSFFTETG (SEQ ID NO:293) [0571] TGFPR2 Intracellular domain

CYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDT LVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFL TAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTP CGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYM APEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHP CVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSE LEHLDRLSGRSCSEEKIPEDGSLNTTK (SEQ ID NO:294) [0572] IL- 1 ORA Intracellular domain

QLYVRRRKKLPSVLLFKKPSPFIFISQRPSPETQDTIHPLDEEAFLKVSPELKNLDLHGSTD SGFGSTKPSLQTEEPQFLLPDPHPQADRTLGNREPPVLGDSCSSGSSNSTDSGICLQEPSL SPSTGPTWEQQVGSNSRGQDDSGIDLVQNSEGRAGDTQGGSALGHHSPPEPEVPGEEDP AAVAFQGYLRQTRCAEEKATKTGCLEEESPLTDGLGPKFGRCLVDEAGLHPPALAKGY LKQDPLEMTL AS SGAPTGQWNQPTEEWSLLALS SC SDLGISDWSF AHDLAPLGC VAAP GGLLGSFNSDLVTLPLISSLQSSE (SEQ ID NO:295) [0573] IL-4RA Intracellular domain

KIKKEWWDQIPNPARSRLVAIIIQDAQGSQWEKRSRGQEPAKCPHWKNCLTKLLPCFLE HNMKRDEDPHKAAKEMPFQGSGKSAWCPVEISKTVLWPESISVVRCVELFEAPVECEE EEEVEEEKGSFCASPESSRDDFQEGREGIVARLTESLFLDLLGEENGGFCQQDMGESCLL PPSGSTSAHMPWDEFPSAGPKEAPPWGKEQPLHLEPSPPASPTQSPDNLTCTETPLVIAG NPAYRSFSNSLSQSPCPRELGPDPLLARHLEEVEPEMPCVPQLSEPTTVPQPEPETWEQIL RRNVLQHGAAAAP VS APTSGYQEFVHAVEQGGTQ AS AVVGLGPPGEAGYKAF S SLL AS S AVSPEKCGFGAS SGEEGYKPFQDLIPGCPGDP APVPVPLFTFGLDREPPRSPQ SSHLPS S SPEHLGLEPGEKVEDMPKPPLPQEQATDPLVDSLGSGIVYSALTCHLCGHLKQCHGQED GGQTPVMASPCCGCCCGDRSSPPTTPLRAPDPSPGGVPLEASLCPASLAPSGISEKSKSSS SFHPAPGNAQSSSQTPKIVNFVSVGPTYMRVS (SEQ ID NO:296) [0574] IL-7RA Intracellular domain

KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFL QDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSS RSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQE EAYVTMSSFYQNQ (SEQ ID NO:297)

[0575] Fas Intracellular domain

KRKEVQKTCRKHRKENQGSHESPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFV RKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTL AEKIQTIILKDITSDSENSNFRNEIQSLV (SEQ ID NO:298) [0576] TRAILR2 Intracellular domain CKSLLWKKVLPYLKGICSGGGGDPERVDRSSQRPGAEDNVLNEIVSILQPTQVPEQEME VQEPAEPTGVNMLSPGESEHLLEPAEAERSQRRRLLVPANEGDPTETLRQCFDDFADLV PFDSWEPLMRI<LGLMDNEII<VAI<AEAAGHRDTLYTMLII<WVNI<TGRDASVHTLLDAL ETLGERLAKQKIEDHLLSSGKFMYLEGNADSAMS (SEQ ID NO:299) [0577] IL12R (IL-12 receptor) subunit beta-1 intracellular domain

NRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGER TEPLEKTELPEGAPELALDTELSLEDGDRCKAKM (SEQ ID NO:313) [0578] IL12R (IL-12 receptor) subunit beta-2 intracellular domain HYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPE PLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQ LVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEE LEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML (SEQ ID NO:314) [0579] IL18R1 (Interleukin- 18 receptor 1) intracellular domain YRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFG YI<LCIFERDVVPGGAVVDEIHSLIEI<SRRLIIVLSI<SYMSNEVRYELESGLHEALVERI<II< IILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRD EPEVLPVLSES (SEQ ID NO:315) [0580] IL21R intracellular domain

SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVP STLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPY GLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGC VSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMD TFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS (SEQ ID NO:316)

[0581] In some embodiments, the costimulatory polypeptide is selected from the group consisting of 0X40, CD2, CD27, CD5, IL12R, IL18R, IL21R, ICAM-1, ICOS (CD278), 4-1BB (CD 137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, FcyRIII, CD3 zeta, CD28, CD27, ICOS, DAP 10, DAP12, LFA-1 (CD1 la/CD18) an MHC class 1 molecule, BTLA and a Toll ligand receptor, lymphocyte function-associated antigen-1 (LFA-1, also known as CDl la/CD18), CD276 (B7- H3), IL-15Ra, and a ligand that specifically binds with CD83. [0582] In some embodiments, the costimulatory polypeptide is IL-15Ra. In some embodiments, the sequence of the switch intracellular domain is from the intracellular domain of IL-15Ra. For example, in some embodiments, the sequence of the switch intracellular domain comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:372. For another example, in some embodiments, the sequence of the switch intracellular domain comprises a sequence or portion thereof of SEQ ID NO:372. In some embodiments, the sequence of the switch intracellular domain comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:383. In some embodiments, the sequence of the switch intracellular domain comprises a sequence or portion thereof of SEQ ID NO: 383.

[0583] In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein. For example, in some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:372 or SEQ ID NO:383. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:372 or SEQ ID NO:383. [0584] In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein. For example, in some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids deleted from the N-terminal or C-terminal end of the sequence of an IL-15Ra intracellular domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids deleted from the N-terminal or C-terminal end of the sequence of an IL-15Ra intracellular domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 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, or 35 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:372. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 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, or 35 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:372. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 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, or 35 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:372. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 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, or 35 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:372. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:383. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:383. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids independently deleted from both N- terminal and C-terminal ends of the sequence of SEQ ID NO: 383. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:383.

Switch Transmembrane Domain

[0585] In some embodiments, the TGFBr2 switch polypeptide further comprises a switch transmembrane domain. In some embodiments, the TGFBr2 extracellular domain or functional fragment thereof is operably linked to the switch intracellular domain via the switch transmembrane domain.

[0586] In some embodiments, the switch transmembrane domain is derived from a TGFBr2 transmembrane domain. In some embodiments, the switch transmembrane domain is a TGFBr2 transmembrane domain. In some embodiments, the switch transmembrane domain comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272. In some embodiments, the switch transmembrane domain comprises the sequence of SEQ ID NO:272. In some embodiments, the sequence of the switch transmembrane domain is a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272. In some embodiments, the sequence of the switch transmembrane domain is the sequence of SEQ ID NO:272.

[0587] In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of a TGFBr2 transmembrane domain as described herein. For example, in some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the sequence of a TGFBr2 transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of a TGFBr2 transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the sequence of SEQ ID NO:272. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:272.

[0588] In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of a TGFBr2 transmembrane domain as described herein. For example, in some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids deleted from the N-terminal or C- terminal end of the sequence of a TGFBr2 transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids deleted from the N-terminal or C-terminal end of the sequence of a TGFBr2 transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:272. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:272. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of a TGFBr2 transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids independently deleted from both N- terminal and C-terminal ends of the sequence of a TGFBr2 transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:272. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:272.

[0589] In some embodiments, the switch transmembrane domain is derived from a transmembrane domain of the costimulatory polypeptide. In some embodiments, the switch transmembrane domain is a transmembrane domain of the costimulatory polypeptide. In some embodiments, the switch transmembrane domain is derived from a transmembrane domain of CD28. In some embodiments, the switch transmembrane domain is derived from a transmembrane domain of 4-1BB. In some embodiments, the switch transmembrane domain is a transmembrane domain of CD28. In some embodiments, the switch transmembrane domain is a transmembrane domain of 4-1BB. In some embodiments, the switch transmembrane domain comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence to SEQ ID NO:275 or SEQ ID NO:279. In some embodiments, the switch transmembrane domain comprises the sequence of SEQ ID NO:275 or SEQ ID NO:279. In some embodiments, the sequence of the switch transmembrane domain is a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence to SEQ ID NO:275 or SEQ ID NO:279. In some embodiments, the sequence of the switch transmembrane domain is the sequence of SEQ ID NO:275 or SEQ ID NO:279.

[0590] In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of a CD28 transmembrane domain or a 4- IBB transmembrane domain as described herein. For example, in some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of a CD28 transmembrane domain or a 4- IBB transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the sequence of a CD28 transmembrane domain or a 4- IBB transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the sequence of SEQ ID NO:275 or SEQ ID NO:279. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:275 or SEQ ID NO:279.

[0591] In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of a CD28 transmembrane domain or a 4-1BB transmembrane domain as described herein. For example, in some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids deleted from the N-terminal or C-terminal end of the sequence of a CD28 transmembrane domain or a 4-1BB transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids deleted from the N-terminal or C-terminal end of the sequence of a CD28 transmembrane domain or a 4-1BB transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:275 or SEQ ID NO:279. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:275 or SEQ ID NO:279. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of a CD28 transmembrane domain or a 4-1BB transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of a CD28 transmembrane domain or a 4-1BB transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:275 or SEQ ID NO:279. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:275 or SEQ ID NO:279.

[0592] In some embodiments, the switch transmembrane domain is derived from a transmembrane domain of IL-15Ra. In some embodiments, the switch transmembrane domain is a transmembrane domain of IL-15Ra. In some embodiments, the switch transmembrane domain comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence to SEQ ID NO:300. In some embodiments, the switch transmembrane domain comprises the sequence of SEQ ID NO:300. In some embodiments, the sequence of the switch transmembrane domain is a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence to SEQ ID NO:300. In some embodiments, the sequence of the switch transmembrane domain is the sequence of SEQ ID NO:300. [0593] In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra transmembrane domain as described herein. For example, in some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the sequence of an IL-15Ra transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:300. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N- terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:300.

[0594] In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra transmembrane domain as described herein. For example, in some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids deleted from the N-terminal or C-terminal end of the sequence of an IL-15Ra transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids deleted from the N-terminal or C-terminal end of the sequence of an IL-15Ra transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids deleted from the N-terminal or C- terminal end of the sequence of SEQ ID NO:300. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9,

10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:300. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of an IL-15Ra transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of an IL- 15Ra transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids independently deleted from both N- terminal and C-terminal ends of the sequence of SEQ ID NO:300. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:300.

[0595] In some embodiments, the switch transmembrane domain is derived from any one selected from the group consisting of an ICOS transmembrane domain or a fragment thereof, a PD-1 transmembrane domain or a fragment thereof, a CTLA4 transmembrane domain or a fragment thereof, a CD200R transmembrane domain or a fragment thereof, a BTLA transmembrane domain or a fragment thereof, a TIM-3 transmembrane domain or a fragment thereof, a TIGIT transmembrane domain or a fragment thereof, a CD28 transmembrane domain or a fragment thereof, a TGFPR2 transmembrane domain or a fragment thereof, a 4-IBB transmembrane domain or a fragment thereof, an IL- 1 ORA transmembrane domain or a fragment thereof, an IL-7RA transmembrane domain or a fragment thereof, an IL-4RA transmembrane domain or a fragment thereof, a Fas transmembrane domain or a fragment thereof, a MyD88 transmembrane domain or a fragment thereof, an TRAIL-R2 transmembrane domain or a fragment thereof, an IL12R transmembrane domain or a fragment thereof, an IL18R transmembrane domain or a fragment thereof, an IL21R transmembrane domain or a fragment thereof, and a combination thereof.

[0596] In some embodiments, an IL12R transmembrane domain or a fragment thereof is an IL- 12 receptor subunit beta-1 transmembrane domain or a fragment thereof. In some embodiments, an IL12R transmembrane domain or a fragment thereof is an IL-12 receptor subunit beta-2 transmembrane domain or a fragment thereof. In some embodiments, an IL18R transmembrane domain or a fragment thereof is an interleukin- 18 receptor 1 transmembrane domain or a fragment thereof.

[0597] In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one selected from SEQ ID NOs:301, 279, 302, 303, 275, 304, 305, 306, 307, 272, 308, 309, 310, 311, 312, 317, 318, 319, 320, and a combination thereof. In some embodiments, the TGFBr2 switch polypeptide comprises any one sequence selected from the group consisting of SEQ ID NOs:301, 279, 302, 303, 275, 304, 305, 306, 307, 272, 308, 309, 310, 311, 312, 317, 318, 319, 320, and a combination thereof. In some embodiments, the sequence of the switch intracellular domain is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one selected from SEQ ID NOs:301, 279, 302, 303, 275, 304, 305, 306, 307, 272, 308, 309, 310, 311, 312, 317, 318, 319, 320, and a combination thereof. In some embodiments, the sequence of the switch intracellular domain is any one sequence selected from the group consisting of SEQ ID NOs:301, 279, 302, 303, 275, 304, 305, 306, 307, 272, 308, 309, 310, 311, 312, 317, 318, 319, 320, and a combination thereof.

[0598] PD -1 Transmembrane domain

VGVVGGLLGSLVLLVWVLAVI (SEQ ID NO:301) [0599] 4- IBB Transmembrane domain

IISFFLALTSTALLFLLFFLTLRFSVV (SEQ ID NO:279)

[0600] ICOS Transmembrane

FWLPIGCAAFVVVCILGCILI (SEQ ID NO: 302) [0601] CTLA4 Transmembrane domain FLLWILAAVSSGLFFYSFLLT (SEQ ID NO:303) [0602] CD28 Transmembrane domain

FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:275)

[0603] CD200R Transmembrane domain

LTGNKSLYIELLPVPGAKKSA (SEQ ID NO: 304)

[0604] BTLA Transmembrane domain

LLPLGGLPLLITTCFCLFCCL (SEQ ID NO: 305)

[0605] TIM-3 Transmembrane domain

IYIGAGICAGLALALIFGALI (SEQ ID NO: 306)

[0606] TIGIT Transmembrane domain

LLGAMAATLVVICTAVIVVVA (SEQ ID NO: 307)

[0607] TGFPR2 Transmembrane domain

VTGISLLPPLGVAISVIIIFY (SEQ ID NO:272)

[0608] IL-10RA Transmembrane domain

VIIFFAFVLLLSGALAYCLAL (SEQ ID NO:308)

[0609] IL-4RA Transmembrane domain

LLLGVSVSCIVILAVCLLCYVSIT (SEQ ID NO: 309)

[0610] IL-7RA Transmembrane domain

PILLTISILSFFSVALLVILACVLW (SEQ ID NO:310)

[0611] Fas Transmembrane domain

LGWLCLLLLPIPLIVWV (SEQ ID NO: 311)

[0612] TRAILR2 Transmembrane domain

LSGIIIGVTVAAVVLIVAVFV (SEQ ID NO:312)

[0613] IL12R subunit beta-1 transmembrane domain

WLIFFASLGSFLSILLVGVLGYLGL (SEQ ID NO:317)

[0614] IL12R subunit beta-2 transmembrane domain

WMAFVAPSICIAIIMVGIFST (SEQ ID NO:318)

[0615] IL18R1 (Interleukin- 18 receptor 1) transmembrane domain

GMIIAVLILVAVVCLVTVCVI (SEQ ID NO:319)

[0616] IL21R transmembrane domain

GWNPHLLLLLLLVIVFIPAFW (SEQ ID NO: 320)

Additional Intracellular Domain

[0617] In some embodiments, the TGFBr2 switch polypeptide further comprises an additional intracellular domain. In some embodiments, the additional intracellular domain is operably linked to the C-terminus of the switch intracellular domain. In some embodiments, the additional intracellular domain is operably linked to the N-terminus of the switch intracellular domain. In some embodiments, the additional intracellular domain is operably linked to the C-terminus or the N-terminus of the switch intracellular domain. In some embodiments, the additional intracellular domain is operably linked to the C-terminus or the N-terminus of the switch intracellular domain via a linker. In some embodiments, the TGFBr2 switch polypeptide may comprise two or more additional intracellular domains. In some embodiments, the additional intracellular domains are operably linked to the C-terminus and the N-terminus of the switch intracellular domain, independently. In some embodiments, the additional intracellular domains are operably linked to the C-terminus, to the N-terminus, or independently to the C-terminus and the N-terminus of the switch intracellular domain via a linker.

[0618] In some embodiments, the TGFBr2 switch polypeptide further comprises one or more additional intracellular domains. In some embodiments, one or more additional intracellular domains are the same intracellular domain. In some embodiments, one or more additional intracellular domains are different intracellular domains.

[0619] In some embodiments, the additional intracellular domain is derived from an intracellular domain of IL-15Ra. In some embodiments, the additional intracellular domain comprises an intracellular domain of IL-15Ra or a fragment thereof.

[0620] In some embodiments, IL-15Ra 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, IL-15Ra or a fragment thereof comprises a sequence having at least about 50%, 55%, 60%, 6%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to a sequence encoding IL-15Ra. In some embodiments, IL-15Ra 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.

[0621] In some embodiments, the additional intracellular domain comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:372 or SEQ ID NO:383. In some embodiments, the additional intracellular domain comprises the sequence of SEQ ID NO:372 or SEQ ID NO:383. In some embodiments, the sequence of the additional intracellular domain is a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:372 or SEQ ID NO:383. In some embodiments, the sequence of the additional intracellular domain is the sequence of SEQ ID NO:372 or SEQ ID NO:383.

[0622] In some embodiments, the additional intracellular domain comprises a sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein. For example, in some embodiments, the additional intracellular domain comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N- terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL- 15Ra intracellular domain as described herein. In some embodiments, the additional intracellular domain comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein. In some embodiments, the additional intracellular domain comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:372 or SEQ ID NO:383. In some embodiments, the additional intracellular domain comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:372 or SEQ ID NO:383.

[0623] In some embodiments, the additional intracellular domain comprises a sequence having a deletion of amino acid residue(s) from the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein. For example, in some embodiments, the additional intracellular domain comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids deleted from the N-terminal or C-terminal end of the sequence of an IL- 15Ra intracellular domain as described herein. In some embodiments, the additional intracellular domain comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids deleted from the N-terminal or C-terminal end of the sequence of an IL-15Ra intracellular domain as described herein. In some embodiments, the additional intracellular domain comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein. In some embodiments, the additional intracellular domain comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein. In some embodiments, the additional intracellular domain comprises a sequence having at least about 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, or 35 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:372. In some embodiments, the additional intracellular domain comprises a sequence having about 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, or 35 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:372. In some embodiments, the additional intracellular domain comprises a sequence having at least about 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, or 35 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:372. In some embodiments, the additional intracellular domain comprises a sequence having about 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, or 35 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:372. In some embodiments, the additional intracellular domain comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO: 383. In some embodiments, the additional intracellular domain comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:383. In some embodiments, the additional intracellular domain comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids independently deleted from both N-terminal and C- terminal ends of the sequence of SEQ ID NO: 383. In some embodiments, the additional intracellular domain comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids independently deleted from both N- terminal and C-terminal ends of the sequence of SEQ ID NO: 383.

Examples of TGFBr2 switch Polypeptides

[0624] In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain derived from a TGFBr2 transmembrane domain and an intracellular signaling domain of 4-1BB.

[0625] In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to any one of the sequences listed in Table 6 or a fragment thereof. In some embodiments, the TGFBr2 switch polypeptide comprises any one of the sequence listed in Table 6 or a fragment thereof. In some embodiments, the sequence of the TGFBr2 switch polypeptide is a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to any one of the sequences listed in Table 6 or a fragment thereof. In some embodiments, the sequence of the TGFBr2 switch polypeptide comprises any one of the sequence listed in Table 6 or a fragment thereof.

[0626] In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272 and a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:277. In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272 operatively linked a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:277. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:272 and the sequence of SEQ ID NO:277. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:272 operatively linked to the sequence of SEQ ID NO:277.

[0627] In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:285. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:285. In some embodiments, the sequence of the TGFBr2 switch polypeptide is a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:285. In some embodiments, the sequence of the TGFBr2 switch polypeptide is the sequence of SEQ ID NO:285.

[0628] In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain derived from a 4- IBB transmembrane domain and an intracellular signaling domain of 4-1BB.

[0629] In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:279 and a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:277. In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:279 operatively linked a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:277. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:279 and the sequence of SEQ ID NO:277. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:279 operatively linked to the sequence of SEQ ID NO:277.

[0630] In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:286. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:286. In some embodiments, the sequence of the TGFBr2 switch polypeptide is a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:286. In some embodiments, the sequence of the TGFBr2 switch polypeptide is the sequence of SEQ ID NO:286.

[0631] In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain derived from a TGFBr2 transmembrane domain and an intracellular signaling domain of CD28.

[0632] In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272 and a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:273. In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272 operatively linked a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:273. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:272 and the sequence of SEQ ID NO:273. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:272 operatively linked to the sequence of SEQ ID NO:273.

[0633] In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:283. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:283. In some embodiments, the sequence of the TGFBr2 switch polypeptide is a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:283. In some embodiments, the sequence of the TGFBr2 switch polypeptide is the sequence of SEQ ID NO:283.

[0634] In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain derived from a CD28 transmembrane domain and an intracellular signaling domain of CD28.

[0635] In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:275 and a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:273. In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:275 operatively linked a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:273. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:275 and the sequence of SEQ ID NO:273. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:275 operatively linked to the sequence of SEQ ID NO:273.

[0636] In some embodiments, the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:284. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:284. In some embodiments, the sequence of the TGFBr2 switch polypeptide is a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:284. In some embodiments, the sequence of the TGFBr2 switch polypeptide is the sequence of SEQ ID NO:284.

Dominant negative TGFBR2 receptors

[0637] Provided herein are recombinant nucleic acids comprising: a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked, and a second nucleic acid sequence encoding C-X-C chemokine receptor type 6 (CXCR6) or a functional fragment thereof. In some embodiments, the recombinant nucleic acids as described herein further comprises a third nucleic acid sequence encoding a dominant negative TGFBR2 receptor or a functional fragment thereof. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a linker. In some embodiments, the linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A cleavage site. In some embodiments, the cleavage site can be a self-cleaving peptide such as a T2A, P2A, E2A or F2A cleavage site. In some embodiments, the 2A cleavage site is a T2A cleavage site or a P2A cleavage site. In some embodiments, the third nucleic acid sequence are operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof. In some embodiments, the third nucleic acid sequence are operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof by a linker. In some embodiments, the third nucleic acid sequence are operatively linked to the first nucleic acid sequence, and/or the second nucleic acid sequence, independently. In some embodiments, the third nucleic acid sequence are operatively linked to the first nucleic acid sequence, and/or the second nucleic acid sequence, independently, by a linker. In some embodiments, the linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A cleavage site. In some embodiments, the 2A cleavage site is a T2A cleavage site or a P2A cleavage site. In some embodiments, the third nucleic acid sequence and the first nucleic acid sequence, the third nucleic acid sequence and the second nucleic acid sequence, or the third nucleic acid sequence, the first nucleic acid sequence, and the second nucleic acid sequence are present on different nucleic acid molecules.

[0638] In some embodiments, the a dominant negative TGFBR2 receptor comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:433 or SEQ ID NO:434. In some embodiments, the dominant negative TGFBR2 receptor comprises the sequence of SEQ ID NO:433 or SEQ ID NO:434. In some embodiments, the sequence of the dominant negative TGFBR2 receptor is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to SEQ ID NO:433 or SEQ ID NO:434. In some embodiments, the sequence of the dominant negative TGFBR2 receptor is the sequence of SEQ ID NO:433 or SEQ ID NO:434.

[0639] In some embodiments, the dominant negative TGFBR2 receptor comprises an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C- terminal ends of a sequence of a dominant negative TGFBR2 receptor as described herein. For example, in some embodiments, the dominant negative TGFBR2 receptor comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N- terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of a dominant negative TGFBR2 receptor as described herein. In some embodiments, the dominant negative TGFBR2 receptor comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of a dominant negative TGFBR2 receptor as described herein. In some embodiments, the dominant negative TGFBR2 receptor comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:433 or SEQ ID NO:434. In some embodiments, the dominant negative TGFBR2 receptor comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N- terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:433 or SEQ ID NO:434.

[0640] In some embodiments, the dominant negative TGFBR2 receptor comprises amino acid residue deletions from the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of a dominant negative TGFBR2 receptor as described herein. For example, in some embodiments, the dominant negative TGFBR2 receptor comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 amino acids deleted from the N-terminal or C-terminal end of a sequence of a dominant negative TGFBR2 receptor as described herein. In some embodiments, the dominant negative TGFBR2 receptor comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 amino acids deleted from the N-terminal or C-terminal end of a sequence of a dominant negative TGFBR2 receptor as described herein. In some embodiments, the dominant negative TGFBR2 receptor comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:433 or SEQ ID NO:434. In some embodiments, the dominant negative TGFBR2 receptor comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:433 or SEQ ID NO:434. In some embodiments, the dominant negative TGFBR2 receptor comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 amino acids independently deleted from both N-terminal and C-terminal ends of a sequence of a dominant negative TGFBR2 receptor as described herein. In some embodiments, the dominant negative TGFBR2 receptor comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 amino acids independently deleted from both N-terminal and C-terminal ends of a sequence of a dominant negative TGFBR2 receptor as described herein. In some embodiments, the dominant negative TGFBR2 receptor comprises a sequence having at least about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:433 or SEQ ID NO:434. In some embodiments, the dominant negative TGFBR2 receptor comprises a sequence having about 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, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 amino acids independently deleted from both N-terminal and C- terminal ends of the sequence of SEQ ID NO:433 or SEQ ID NO:434.

[0641] In some embodiments, the a dominant negative TGFBR2 receptor comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one of the sequences listed in Table 7. In some embodiments, the dominant negative TGFBR2 receptor comprises any one of the sequences listed in Table 7. In some embodiments, the sequence of the dominant negative TGFBR2 receptor is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to any one of the sequences listed in Table 7. In some embodiments, the sequence of the dominant negative TGFBR2 receptor is any one of the sequences listed in Table 7.

[0642] An exemplary dominant negative TGFBR2 receptor with a signal peptide: MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAAS PKCIMKEKKKPGETFFMCSC S SDECNDNIIF SEEYNTSNPDLLL VIFQ VTGISLLPPLGVAI SVIIIFYCYRVNRQQKLSS (SEQ ID NO:433)

[0643] An exemplary dominant negative TGFBR2 receptor without a signal peptide: TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQE VCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSS (SEQ ID NO:434)

PD-1 Switch Molecule

[0644] In some embodiments, the modified cells, e.g., 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, 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. In some embodiments, a T cell expressing the TFP as described herein, CXCR6 or a functional fragment thereof as descried herein, and a PD-1 switch molecule as descried herein can inhibit tumor growth when expressed in a T cell.

[0645] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first sequence encoding a TFP as described herein, a second sequence encoding CXCR6 or a functional fragment thereof as descried herein, and a third nucleic acid sequence encoding an agent that can enhance the activity of a modified T cell expressing the TFP as described herein and CXCR6 or a functional fragment thereof as descried herein. In some embodiments, the third nucleic acid sequence is included in a separate nucleic acid sequence from the recombinant nucleic acid molecules. In some embodiments, the third nucleic acid sequence is included in the same nucleic acid molecule as the recombinant nucleic acid molecules. For example, in one embodiment, the agent that can enhance the activity of a modified T cell can be a PD-1 polypeptide. In these embodiments, the PD-1 polypeptide may be operably linked to the N-terminus of an intracellular domain of a costimulatory polypeptide via the C-terminus of the PD-1 polypeptide. For example, in another embodiment, the agent that can enhance the activity of a modified T cell can be an anti -PD-1 antibody, or antigen binding fragment thereof. In this embodiment, the anti-PD-1 antibody or antigen binding fragment thereof may be operably linked to the N-terminus of an intracellular domain of a costimulatory polypeptide via the C-terminus of the anti-PD-1 antibody, or antigen binding fragment thereof. In some embodiments, the PD-1 polypeptide or anti-PD-1 antibody is linked to the intracellular domain of the costimulatory polypeptide via the transmembrane domain of PD-1. In some embodiments, the costimulatory polypeptide is selected from the group consisting of 0X40, CD2, CD27, CD5, ICAM-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, IL- 15Ra, IL12R, IL18R, IL21R, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, CD226, FcyRI, FcyRII, and FcyRIII. In some embodiments, the costimulatory peptide is CD28.

[0646] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first sequence encoding a TFP as described herein and a second sequence encoding CXCR6 or a functional fragment thereof as descried herein, wherein the recombinant nucleic acid molecules further comprising an agent that can enhance the activity of a modified T cell expressing the TFP as described herein and CXCR6 or a functional fragment thereof as descried herein. In another aspect, the cells expressing TFP as described herein and CXCR6 or a functional fragment thereof as descried herein can further express another agent, e.g., an agent which enhances the activity of a modified T cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., PD-1, can, in some embodiments, decrease the ability of a modified cell, e.g., T cell, to mount an immune effector response. Examples of inhibitory molecules include PD-1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 and TGFR beta. In one embodiment, the agent which inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain as described herein. In one embodiment, the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD-1, LAG3, CTLA4, CD 160, BTLA, LAIR1, TIM3, 2B4, and TIGIT, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD-1 or a fragment thereof (e.g., at least a portion of an extracellular domain of PD-1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein). In one embodiment, the agent that inhibits an inhibitory molecule is a fusion protein that comprises an extracellular domain having an antibody or antibody fragment that specifically binds PD-1, e.g., an anti-PD-1 antibody described herein, and further comprises a transmembrane domain.

Suitable transmembrane domains for use with the present disclosure include, but are not limited to, transmembrane domains derived from CD28, CD3s, CD3(^, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD41, CD64, CD68, CD80, CD86, CD134, CD137, CD154, ICOS, 4- IBB, 0X40, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the fusion protein further comprises a co-stimulatory domain. Suitable co-stimulatory domains for use with the pre-sent disclosure include, but are not limited to, co-stimulatory domains derived from CD27, CD28, 4- 1BB (CD137), 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, PD- 1, CD258, ICAM, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the recombinant nucleic acid molecules as described herein further comprises a sequence encoding PD-1 or a fragment thereof. In some embodiments, the recombinant nucleic acid molecules as described herein further comprises a sequence encoding the extracellular domain of PD-1. In some embodiments, the recombinant nucleic acid molecules as described herein comprises a sequence encoding the extracellular domain and transmembrane domain of PD-1. In some embodiments, the recombinant nucleic acid molecules as described herein may further comprise a sequence encoding CD28 or a fragment thereof. In some embodiments, the recombinant nucleic acid molecules as described herein comprises a sequence encoding the intracellular domain of CD28. In some embodiments, the recombinant nucleic acid molecules as described herein comprises a sequence encoding a fusion protein comprising the PD-1 extracellular domain and transmembrane domain linked to the CD28 intracellular domain linked to intracellular domain. In some embodiments, the agent comprises the extracellular and transmembrane domain of PD-1 fused to the intracellular signaling domain of CD28. In some embodiments, the agent comprises SEQ ID NO: 1239 or SEQ ID NO: 1244. PD1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al., 1996, Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2, have been shown to downregulate T cell activation upon binding to PD1 (Freeman et al., 2000 J. Exp. Med. 192: 1027-34; Latchman et al., 2001 Nat. Immunol. 2:261-8; Carter et al., 2002 Eur. J. Immunol. 32:634-43). PD-L1 is abundant in human cancers (Dong et al., 2003 J. Mol. Med. 81 :281-7; Blank et al., 2005 Cancer Immunol. Immunother. 54:307-314; Konishi et al., 2004 Clin. Cancer Res. 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.

[0647] In one embodiment, the agent comprises the extracellular domain (ECD) of an inhibitory molecule, e.g., PD-1 can be fused to a transmembrane domain and optionally an intracellular signaling domain such as 41BB and CD3 zeta (also referred to herein as a PD-1 TFP). In one embodiment, the PD-1 TFP, when used in combinations with an anti-TAA TFP as described herein and CXCR6 or a functional fragment thereof as descried herein, improves the persistence of the T cell. In one embodiment, the TFP is a PD-1 TFP comprising the extracellular domain of PD-1. Alternatively, provided are TFPs containing an antibody or antibody fragment such as a scFv that specifically binds to the Programmed Death-Ligand 1 (PD-L1) or Programmed Death- Ligand 2 (PD-L2).

[0648] In another aspect, the present disclosure provides a population of TFP-expressing T cells, e.g., TFP-T cells co-expressing CXCR6 or a functional fragment thereof as described herein. In some embodiments, the population of TFP-expressing T cells co-expressing CXCR6 or a functional fragment thereof as described herein comprises a mixture of cells expressing different TFPs. For example, in one embodiment, the population of TFP-T cells co-expressing CXCR6 or a functional fragment thereof as described herein can include a first cell expressing a TFP having a binding domain described herein, and a second cell expressing a TFP having a different anti-TAA binding domain, e.g., a binding domain described herein that differs from the binding domain in the TFP expressed by the first cell. As another example, the population of TFP- expressing cells co-expressing CXCR6 or a functional fragment thereof as described herein can include a first cell expressing a TFP that includes a first binding domain binding domain, e.g., as described herein, and a second cell expressing a TFP that includes an antigen binding domain to a target other than the binding domain of the first cell (e.g., another tumor-associated antigen). [0649] In one aspect, the present disclosure provides a population of cells wherein at least one cell in the population expresses a TFP having a domain as described herein and CXCR6 or a functional fragment thereof as descried herein, and a second cell expressing another agent, e.g., an agent which enhances the activity of a modified T cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., can, in some embodiments, decrease the ability of a modified T cell to mount an immune effector response. Examples of inhibitory molecules include PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, and 2B4. In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In some embodiments, the agent is a cytokine. In some embodiments, the cytokine is IL-15. In some embodiments, IL-15 increases the persistence of the T cells described herein.

Recombinant Nucleic Acid Encoding a PD-1 Switch Molecule

[0650] 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 CXCR6 or a functional fragment thereof as descried herein, and a third nucleic acid sequence encoding a PD-1 switch molecule as described herein. In some embodiments, recombinant nucleic acid molecules comprise a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as descried herein, and a third nucleic acid sequence encoding an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain. In some embodiments, recombinant nucleic acid molecules comprise a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as descried herein, and a third nucleic acid sequence encoding a inhibitory molecule comprising the first polypeptide comprising at least a portion of PD-1 and the second polypeptide comprising a costimulatory domain and primary signaling domain. In some embodiments, a T cell expressing the TFP as descried herein, CXCR6 or a functional fragment thereof as descried herein, and a PD-1 switch molecule as descried herein can inhibit tumor growth when expressed in a T cell.

IL-15 and IL-15 receptor alpha polypeptides

[0651] In some aspects, the TFP-expressing cells that co-express CXCR6 or a functional fragment thereof as described herein can further express another agent, for example, an agent that can enhance longevity or activity of TFP-expressing cells that co-express CXCR6 or a functional fragment thereof as 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 IL-15 and/or interleukin- 15 receptor (IL-15R). In some embodiments, the IL-15R is an IL-15R alpha (IL- 15Ra) subunit.

[0652] The present disclosure encompasses recombinant nucleic acid molecules encoding an 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%, 99.5%, 99.7%, 99.9% 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. [0653] 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: 1245. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 1246. 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: 1245. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise any one of the sequence listed in Table 9 or a fragment thereof. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 1242. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise amino acids 1-162 of SEQ ID NO: 1245. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 1246 and a sequence of SEQ ID NO: 1242. In some embodiments, IL-15 polypeptide is secreted when expressed in a cell, such as a T cell.

[0654] The present disclosure further encompasses recombinant nucleic acid molecules encoding an 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-2RP” 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 subunit 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%, 99.5%, 99.7%, 99.9% 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.

[0655] 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: 1247. 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: 1247.

[0656] 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: 1247. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 1250. [0657] 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: 1247. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 1248.

[0658] 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: 1247. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 1250. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 1251. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 96-267 of SEQ ID NO: 1247. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 1250 and a sequence of SEQ ID NO: 1251.

[0659] 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: 1247. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO: 1249.

[0660] 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: 1243.

[0661] SG3(SG₄)3SG₃SLQ Linker

SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 1243)

[0662] 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: 1245. In some embodiments, the fusion protein may comprise any one of the sequence listed in Table 9 or a fragment thereof. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO: 1242. 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: 1245. In some embodiments, the fusion protein does not comprise a sequence of SEQ ID NO: 1246.

[0663] 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: 1247. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO: 1250.

[0664] 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: 1247. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO: 1248.

[0665] 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: 1247. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO: 1249.

[0666] 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: 1247. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO: 1251.

[0667] 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: 1247. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO: 1250 and a sequence of SEQ ID NO: 1251.

[0668] 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), P-galactosidase (P-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. In some embodiments, the fusion protein further comprises a sequence of SEQ ID NO: 1255.

[0669] Flag x3

DYKDDDDKDYKDDDDKDYKDDDDK (SEQ ID NO: 1255)

[0670] In some embodiments, the fusion protein is expressed on cell surface when expressed in a cell, e.g., a T cell. In some embodiments, the fusion protein is secreted when expressed in a cell, e.g., a T cell.

[0671] In some aspects, cells expressing TFPs, CXCR6 or a functional fragment thereof as descried herein, an IL- 15 polypeptide or a fragment thereof, an IL-15Ra polypeptide or a fragment thereof, and/or a fusion protein comprising an IL-15 polypeptide and an IL-15Ra polypeptide described herein can yet further express another agent that can enhance the activity of a modified T cell expressing TFPs and CXCR6 or a functional fragment thereof as descried herein. For example, in one embodiment, the agent that can enhance the activity of a modified T cell can be a PD-1 polypeptide. In these embodiments, the PD-1 polypeptide may be operably linked to the N-terminus of an intracellular domain of a costimulatory polypeptide via the C- terminus of the PD-1 polypeptide. For example, in another embodiment, the agent that can enhance the activity of a modified T cell expressing TFPs can be an anti -PD-1 antibody, or antigen binding fragment thereof. In this embodiment, the anti-PD-1 antibody or antigen binding fragment thereof may be operably linked to the N-terminus of an intracellular domain of a costimulatory polypeptide via the C-terminus of the anti-PD-1 antibody, or antigen binding fragment thereof. In some embodiments, the PD-1 polypeptide or anti-PD-1 antibody is linked to the intracellular domain of the costimulatory polypeptide via the transmembrane domain of PD- 1. In some embodiments, the costimulatory polypeptide is selected from the group consisting of 0X40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, and FcyRIII. In some embodiments, the costimulatory polypeptide is CD28.

[0672] In some aspects, the agent that can enhance the activity of a modified T cell expressing TFPs and CXCR6 or a functional fragment thereof as descried herein can be linked to an IL- 15Ra polypeptide or a fragment thereof. 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 and CXCR6 or a functional fragment thereof as descried herein to mount an immune effector response. In some embodiments, the agent which inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent may comprise a first polypeptide, e.g., of an inhibitory molecule such as PD-1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4, and TIGIT, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain as described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27, or CD28, as described herein)) and/or a primary signaling domain (e.g., IL- 15Ra described herein). In some embodiments, the agent may be PD-1 or a fragment thereof. For example, the agent may comprise the extracellular domain of PD-1. In some embodiments, the agent may comprise the extracellular domain and transmembrane domain of PD-1. In some embodiments, the agent may further comprise CD28 or a fragment thereof. In some embodiments, the agent may comprise the intracellular domain of CD28. In some embodiments, the agent may comprise a fusion protein comprising the PD-1 extracellular domain and transmembrane domain linked to the CD28 intracellular domain linked to IL-15Ra. In some embodiments, the CD28 intracellular domain is linked to the intracellular domain of IL-15Ra. [0673] In some embodiments, the PD-1 or a fragment thereof may comprise any one of the sequence listed in Table 8 or a fragment thereof. In some embodiments, the PD-1 or a fragment thereof may comprise a sequence of SEQ ID NO: 1256. In some embodiments, the PD-1 or a fragment thereof may comprise a sequence of SEQ ID NO: 1257. In some embodiments, the PD- 1 or a fragment thereof may comprise a sequence of SEQ ID NO: 1258. In some embodiments, the PD-1 or a fragment thereof may comprise a sequence of SEQ ID NO: 1259. In some embodiments, the transmembrane domain of PD-1 may comprise a sequence of SEQ ID NO: 1239 or SEQ ID NO: 1244. In some embodiments, the intracellular domain of CD28 may comprise a sequence of SEQ ID NO: 1260.

[0674] In some aspects, the agent that can enhance the activity of a modified T cell expressing TFPs and CXCR6 or a functional fragment thereof as descried herein can be linked to a fusion protein comprising an IL-15 polypeptide and an IL-15Ra polypeptide. In some embodiments, the agent may be PD-1 or a fragment thereof. For example, the agent may comprise the extracellular domain of PD-1. In some embodiments, the agent may comprise the extracellular domain and transmembrane domain of PD-1. In some embodiments, the agent may further comprise CD28 or a fragment thereof. In some embodiments, the agent may comprise the intracellular domain of CD28. In some embodiments, the agent may comprise a fusion protein comprising the PD-1 extracellular domain and transmembrane domain linked to the CD28 intracellular domain linked to the fusion protein comprising an IL-15 polypeptide and an IL- 15Ra polypeptide. In some embodiments, the CD28 intracellular domain is linked to the intracellular domain of IL-15Ra. In some embodiments, the intracellular domain of IL-15Ra is linked to the IL-15 polypeptide by a linker described herein. In some embodiments, the linker comprises a cleavage site. The cleavage site can be a self-cleaving peptide such as a T2A, P2A, E2A or F2A cleavage site. In some embodiments, the cleavage site can comprise a sequence of SEQ ID NO: 1261 (P2A: GSGATNFSLLKQAGDVEENPG).

[0675] In some embodiments, the fusion protein may comprise a PD-1 or a fragment thereof comprising any one of the sequence listed in Table 8 or a fragment thereof. In some embodiments, the fusion protein may comprise a PD-1 or a fragment thereof comprising a sequence of SEQ ID NO: 1256. In some embodiments, the fusion protein may comprise a PD-1 or a fragment thereof comprising a sequence of SEQ ID NO: 1257. In some embodiments, the fusion protein may comprise a PD-1 or a fragment thereof comprising a sequence of SEQ ID NO: 1258. In some embodiments, the fusion protein may comprise a PD-1 or a fragment thereof comprising a sequence of SEQ ID NO: 1259. In some embodiments, the fusion protein may comprise a PD-1 or a fragment thereof comprising a transmembrane domain of PD-1 comprising a sequence of SEQ ID NO: 1239 or SEQ ID NO: 1244. In some embodiments, the fusion protein may comprise a CD28 or a fragment comprising the intracellular domain of CD28 comprising a sequence of SEQ ID NO: 1260.

[0676] In some embodiments, the intracellular domain of IL-15Ra comprises amino acids 229- 267 of IL-15Ra. In some embodiments, the intracellular domain of IL-15Ra comprises amino acids 229-267 of a sequence of SEQ ID NO: 1247. In some embodiments, the fusion protein comprises a sequence of SEQ ID NO: 1248. In some embodiments, the IL-15 polypeptide comprises IL-15 signal peptide. In some embodiments, the IL-15 polypeptide comprises amino acids 1-29 of IL-15. In some embodiments, the IL-15 polypeptide comprises amino acids 1-29 of a sequence of SEQ ID NO: 1245. In some embodiments, the IL-15 polypeptide comprises a sequence of SEQ ID NO: 1246. In some embodiments, the IL-15 polypeptide comprises amino acids 30-162 of IL-15. In some embodiments, the IL-15 polypeptide comprises amino acids 30- 162 of a sequence of SEQ ID NO: 1245. In some embodiments, the IL-15 polypeptide comprises a sequence of SEQ ID NO: 1242. [0677] Disclosed herein, in some embodiments, are polypeptides encoded by any of recombinant nucleic acid molecules described herein.

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

[0678] Disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein. Disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third nucleic acid sequence encoding an Interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof as described herein. Also disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third 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 as described herein. Further disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third nucleic acid sequence encoding a fusion protein comprising a fusion protein comprising an IL-15Ra polypeptide or a fragment thereof linked to PD-1 or a fragment thereof and/or CD28 or a fragment thereof as described herein.

[0679] Disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof as described herein. Any recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein and a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein may further comprise a third nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof as described herein. Further disclosed herein are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third nucleic acid sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein. Any recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein and a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein may further comprise a third nucleic acid sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein.

[0680] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof as described herein, wherein the third nucleic acid sequence are included in a separate nucleic acid molecule from the first nucleic acid sequence and/or the second nucleic acid sequence. Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof as described herein, wherein the first nucleic acid sequence, the second nucleic acid sequence, and the third 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, the first nucleic acid sequence and the third nucleic acid sequence, and/or the second nucleic acid sequence and the third nucleic acid sequence are operatively linked by a linker. Further disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third nucleic acid sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein, wherein the third nucleic acid sequence is included in a separate nucleic acid molecule from the first nucleic acid sequence and the second nucleic acid sequence. Further disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third nucleic acid sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein, wherein the first nucleic acid sequence, the second nucleic acid sequence, and the third 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, the first nucleic acid sequence and the third nucleic acid sequence, and/or the second nucleic acid sequence and the third nucleic acid sequence are operatively linked by a linker. For example, the linker may be a cleavable linker. In some embodiments, the linker may comprise a protease cleavage site. The cleavage site can be a self-cleaving peptide, for example, a 2A cleavage site such as a T2A, P2A, E2A or F2A cleavage site. In some embodiments, the protease cleavage site is a T2A cleavage site. The cleavage site can comprise a sequence of SEQ ID NO: 1238, when expressed. In some embodiments, the linker comprises a sequence of SEQ ID NO: 1238, when expressed.

[0681] 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: 1245, when expressed. In some embodiments, IL-15 signal peptide comprises a sequence of SEQ ID NO: 1246, 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: 1245. In some embodiments, the nucleic acid sequence encoding the IL-15 polypeptide, or a fragment thereof may comprise a sequence encoding any one of the sequence listed in Table 9 or a fragment thereof. 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: 1242. 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: 1245. 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: 1246 and a sequence of SEQ ID NO: 1242. In some embodiments, the IL-15 polypeptide or a fragment thereof is secreted when expressed in a cell, e.g., a T cell. In some embodiments, the IL-15 polypeptide comprises a sequence of SEQ ID NO: 1242, when expressed.

[0682] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof and an IL-15R subunit or a fragment thereof as described herein, wherein the third nucleic sequence is included in a separate nucleic acid molecule from the first nucleic acid sequence and the second nucleic acid sequence. Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof and an IL-15R subunit or a fragment thereof as described herein, wherein the first nucleic acid sequence, the second nucleic acid sequence, and third 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, the first nucleic acid sequence and the third nucleic acid sequence, and/or the second nucleic acid sequence and the third nucleic acid sequence are operatively linked by a linker as described herein. An IL-15R subunit may be an IL-15R alpha (IL-15Ra), an IL-2R beta (IL-2P), or an IL-2R gamma/the common gamma chain (ZL-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: 1243.

[0683] 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: 1247. 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: 1248.

[0684] 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-15Ra polypeptide or a fragment thereof may comprise a sequence encoding amino acids 31-95 of SEQ ID NO: 1247. 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: 1250.

[0685] 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: 1247. 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:1251.

[0686] 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: 1247. 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: 1250 and a sequence of SEQ ID NO: 1251.

[0687] 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: 1247. 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: 1249.

[0688] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third nucleic acid sequence encoding a fusion protein comprising an IL-15 polypeptide linked to an IL-15Ra subunit as described herein, wherein the third nucleic acid sequence is included in a separate nucleic acid molecule from the first nucleic acid sequence and the second nucleic acid sequence. Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third nucleic acid sequence encoding a fusion protein comprising an IL-15 polypeptide linked to an IL-15Ra subunit as described herein, wherein the first nucleic acid sequence, the second nucleic acid sequence, and the third 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, the first nucleic acid sequence and the third nucleic acid sequence, and/or the first nucleic acid sequence and the third nucleic acid sequence are operatively linked by a first linker as 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. [0689] 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: 1245. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 1246. 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 the fusion protein may comprise a sequence encoding amino acids SO- 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: 1245. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding any one of the sequence listed in Table 9 or a fragment thereof. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 1242. 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: 1245. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 1246 and a sequence encoding a sequence of SEQ ID NO: 1242.

[0690] 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: 1247. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 1248.

[0691] 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: 1247. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 1250.

[0692] 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: 1247. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 1251.

[0693] 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: 1247. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 1250 and a sequence of SEQ ID NO: 1251.

[0694] 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: 1247. In some embodiments, the nucleic acid sequence encoding the fusion protein may comprise a sequence encoding a sequence of SEQ ID NO: 1249.

[0695] 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), P-galactosidase (P-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. In some embodiments, the nucleic acid sequence encoding the fusion protein further comprises a sequence encoding a sequence of SEQ ID NO: 1255.

[0696] In some embodiments, the fusion protein is expressed on cell surface when expressed from the recombinant nucleic acid molecule described herein in a cell, e.g., a T cell. In some embodiments, the fusion protein is secreted when expressed from the recombinant nucleic acid molecule described herein in a cell, e.g., a T cell.

[0697] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, a third nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof as described herein, and a fourth nucleic acid sequence encoding an agent that can enhance the activity of a modified T cell expressing the TFP and CXCR6 or a functional fragment thereof as described herein. In some embodiments, the fourth nucleic acid sequence is included in a separate nucleic acid sequence. In some embodiments, the fourth nucleic acid sequence is included in the same nucleic acid molecule as the first nucleic acid sequence, the second nucleic acid sequence, or the third nucleic acid sequence, or the first, the second, and the third nucleic acid sequences. For example, in one embodiment, the agent that can enhance the activity of a modified T cell can be a PD-1 polypeptide. In these embodiments, the PD-1 polypeptide may be operably linked to the N-terminus of an intracellular domain of a costimulatory polypeptide via the C-terminus of the PD-1 polypeptide. For example, in another embodiment, the agent that can enhance the activity of a modified T cell can be an anti-PD-1 antibody, or antigen binding fragment thereof. In this embodiment, the anti-PD-1 antibody or antigen binding fragment thereof may be operably linked to the N-terminus of an intracellular domain of a costimulatory polypeptide via the C- terminus of the anti-PD-1 antibody, or antigen binding fragment thereof. In some embodiments, the PD-1 polypeptide or anti-PD-1 antibody is linked to the intracellular domain of the costimulatory polypeptide via the transmembrane domain of PD-1. In some embodiments, the costimulatory polypeptide is selected from the group consisting of 0X40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, and FcyRIII.

[0698] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and a third nucleic acid sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein, wherein the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are operatively linked by a first linker as described herein, and wherein the third nucleic acid sequence further encodes an agent that can enhance the activity of a modified T cell expressing the TFP and CXCR6 or a functional fragment thereof. 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 and CXCR6 or a functional fragment thereof to mount an immune effector response. In some embodiments, the agent which inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent may comprise a first polypeptide, e.g., of an inhibitory molecule such as PD-1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4, and TIGIT, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27, or CD28, as described herein)) and/or a primary signaling domain (e.g., IL-15Ra described herein). In some embodiments, the third nucleic acid sequence further comprises a sequence encoding PD-1 or a fragment thereof. In some embodiments, the third nucleic acid sequence comprises a sequence encoding the extracellular domain of PD-1. In some embodiments, the third nucleic acid sequence comprises a sequence encoding the extracellular domain and transmembrane domain of PD-1. In some embodiments, the third nucleic acid sequence may further comprise a sequence encoding CD28 or a fragment thereof. In some embodiments, the third nucleic acid sequence comprises a sequence encoding the intracellular domain of CD28. In some embodiments, the third nucleic acid sequence comprises a sequence encoding a fusion protein comprising the PD-1 extracellular domain and transmembrane domain linked to the CD28 intracellular domain linked to IL-15Ra. In some embodiments, the CD28 intracellular domain is linked to the intracellular domain of IL- 15Ra. In some embodiments, the intracellular domain of IL-15Ra comprises amino acids 229- 267 of IL-15Ra. In some embodiments, the intracellular domain of IL-15Ra comprises amino acids 229-267 of SEQ ID NO: 1247. In some embodiments the intracellular domain of IL-15Ra comprises a sequence of SEQ ID NO: 1248.

[0699] In some embodiments, the third nucleic acid sequence encoding PD-1, or a fragment thereof may comprise a nucleic acid sequence encoding any one of the sequence listed in Table 8 or a fragment thereof. In some embodiments, the third nucleic acid sequence encoding PD-1, or a fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 1256. In some embodiments, the third nucleic acid sequence encoding PD-1, or a fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 1257. In some embodiments, the third nucleic acid sequence encoding PD-1, or a fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 1258. In some embodiments, the third nucleic acid sequence encoding PD-1, or a fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 1259. In some embodiments, the nucleic acid sequence encoding the transmembrane domain of PD-1 may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 1239 or SEQ ID NO: 1244. In some embodiments, the nucleic acid sequence encoding the intracellular domain of CD28 may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 1260. In some embodiments, the intracellular domain of IL-15Ra comprises amino acids 229-267 of IL-15Ra. In some embodiments, the nucleic acid encoding the intracellular domain of IL-15Ra comprises a nucleic acid encoding amino acids 229-267 of SEQ ID NO: 1247. In some embodiments, the nucleic acid encoding the intracellular domain of IL-15Ra comprises a nucleic acid encoding a sequence of SEQ ID NO: 1248.

[0700] Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, a third nucleic acid sequence encoding an IL-15Ra polypeptide or a fragment thereof as described herein and an agent that can enhance the activity of a modified T cell expressing the TFP and CXCR6 or a functional fragment thereof as described herein, and a fourth nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof as described herein. In some embodiments, the third nucleic sequence is included in a separate nucleic acid sequence from the first nucleic acid sequence and the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence, the first nucleic acid sequence and the third nucleic acid sequence, the second nucleic acid sequence and the second nucleic acid sequence, or the first nucleic acid sequence, the second nucleic acid sequence, and the third 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, the first nucleic acid sequence and the third nucleic acid sequence, and/or the second nucleic acid sequence and the third nucleic acid sequence are operatively linked by a first linker as described herein. In some embodiments, the fourth nucleic acid sequence is included in a separate nucleic acid sequence. In some embodiments, the fourth nucleic acid sequence is included in the same nucleic acid molecule as the first nucleic acid sequence, the second nucleic acid sequence, or the third nucleic acid sequence, or the first and the second nucleic acid sequences, the first and the third nucleic acid sequences, the second and the third nucleic acid sequences, or the first, the second, and the third nucleic acid sequences. In some embodiments, the fourth nucleic acid sequence encoding the IL- 15 polypeptide may comprise a sequence encoding amino acids 1-29 of IL-15. In some embodiments, the fourth nucleic acid sequence encoding the IL-15 polypeptide may comprise a sequence encoding amino acids 1-29 of SEQ ID NO: 1245. In some embodiments, the fourth nucleic acid sequence encoding the IL-15 polypeptide may comprise a sequence encoding a sequence of SEQ ID NO: 1246. In some embodiments, the fourth nucleic acid sequence encoding the IL-15 polypeptide may comprise a sequence encoding amino acids 30-162 of IL-15. In some embodiments, the fourth nucleic acid sequence encoding the IL-15 polypeptide may comprise a sequence encoding amino acids 30-162 of SEQ ID NO: 1245. In some embodiments, the fourth nucleic acid sequence encoding the IL-15 polypeptide may comprise a sequence encoding any one of the sequence listed in Table 9 or a fragment thereof. In some embodiments, the fourth nucleic acid sequence encoding the IL-15 polypeptide may comprise a sequence encoding a sequence of SEQ ID NO: 1242. In some embodiments, the IL-15 polypeptide is secreted when expressed in a T cell. In some embodiments, the fourth nucleic acid sequence encoding the IL- 15 polypeptide may comprise a sequence encoding amino acids 1-162 of IL-15. In some embodiments, the fourth nucleic acid sequence encoding the IL-15 polypeptide may comprise a sequence encoding amino acids 1-162 of SEQ ID NO: 1245. In some embodiments, the fourth nucleic acid sequence encoding the IL-15 polypeptide may comprise a sequence encoding a sequence of SEQ ID NO: 1246 and a sequence of SEQ ID NO: 1242.

Combination of Multiple Agents

[0701] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a nucleic acid sequence encoding a TFP as described herein, a nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and one or more of a nucleic acid sequence encoding a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof as described herein, a nucleic acid sequence encoding a dominant negative TGFBR2 receptor or a fragment thereof as described herein, a nucleic acid sequence encoding a PD-1 switch molecule comprising a PD-1 polypeptide or a fragment thereof as described herein, a nucleic acid sequence encoding a CD28 polypeptide or a fragment thereof as described herein, a nucleic acid sequence encoding a PD-1 polypeptide or a fragment thereof fused to a CD28 polypeptide or a fragment thereof fused to an IL-15Ra subunit or a fragment thereof as 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 subunit or a fragment thereof as described herein. [0702] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, a third nucleic acid sequence selected from a nucleic acid sequence encoding a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof as described herein, a nucleic acid sequence encoding a dominant negative TGFBR2 receptor or a fragment thereof as described herein, a nucleic acid sequence encoding a PD-1 switch molecule comprising a PD- 1 polypeptide or a fragment thereof as described herein, a nucleic acid sequence encoding a CD28 polypeptide or a fragment thereof as described herein, a nucleic acid sequence encoding a PD-1 polypeptide or a fragment thereof fused to a CD28 polypeptide or a fragment thereof fused to an IL-15Ra subunit or a fragment thereof as 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 subunit or a fragment thereof as described herein.

[0703] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, a third nucleic acid sequence and a fourth nucleic acid sequence independently selected from a nucleic acid sequence encoding a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof as described herein, a nucleic acid sequence encoding a dominant negative TGFBR2 receptor or a fragment thereof as described herein, a nucleic acid sequence encoding a PD-1 switch molecule comprising a PD-1 polypeptide or a fragment thereof as described herein, a nucleic acid sequence encoding a CD28 polypeptide or a fragment thereof as described herein, a nucleic acid sequence encoding a PD-1 polypeptide or a fragment thereof fused to a CD28 polypeptide or a fragment thereof fused to an IL-15Ra subunit or a fragment thereof as 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 subunit or a fragment thereof as described herein, wherein the third nucleic acid sequence and the fourth nucleic acid sequence are not same.

[0704] In some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as descried herein, and a third nucleic acid sequence encoding any one of a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof, a dominant negative TGFBR2 receptor or a fragment thereof as described herein, an IL-15 polypeptide or a fragment thereof, an IL-15 polypeptide or a fragment thereof operatively linked to an IL-15R subunit or a fragment thereof, a PD-1 polypeptide or a fragment thereof, or a fusion protein comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15Ra as described herein. Disclosed herein, in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as descried herein, a third nucleic acid sequence encoding any one of a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof, a dominant negative TGFBR2 receptor or a fragment thereof as described herein, an IL-15 polypeptide or a fragment thereof, an IL-15 polypeptide or a fragment thereof operatively linked to an IL-15R subunit or a fragment thereof, a PD-1 polypeptide or a fragment thereof, or a fusion protein comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL- 15Ra as described herein, and a fourth nucleic acid sequence encoding any one of a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof, a dominant negative TGFBR2 receptor or a fragment thereof as described herein, an IL-15 polypeptide or a fragment thereof, an IL- 15 polypeptide or a fragment thereof operatively linked to an IL-15R subunit or a fragment thereof, a PD-1 polypeptide or a fragment thereof, or a fusion protein comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15Ra as described herein, wherein the third sequence and the fourth sequence do not encode the same polypeptide.

[0705] 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: 195. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a sequence encoding CD3s. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO: 196. In some embodiments, CXCR6 or a functional fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence listed in Table 4 or a fragment thereof.

[0706] In some embodiments, CXCR6 or a functional fragment thereof comprises any one sequence listed in Table 4 or a fragment thereof. In some embodiments, CXCR6 or a functional fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID N0s:400-402. In some embodiments, CXCR6 or a functional fragment thereof comprises any one sequence selected from SEQ ID N0s:400-402. In some embodiments, a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence listed in Table 6 or a fragment thereof. In some embodiments, a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof comprises any one sequence listed in Table 6 or a fragment thereof. In some embodiments, a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 283, 284, 285, and 286. In some embodiments, a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof comprises any one sequence selected from SEQ ID NOs: 283, 284, 285, and 286. In some embodiments, a dominant negative TGFBR2 receptor or a fragment thereof as described herein comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence listed in Table 7 or a fragment thereof. In some embodiments, a dominant negative TGFBR2 receptor or a fragment thereof as described herein comprises any one sequence listed in Table 7 or a fragment thereof. In some embodiments, a dominant negative TGFBR2 receptor or a fragment thereof as described herein comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NO:433 and SEQ ID NO:434. In some embodiments, a dominant negative TGFBR2 receptor or a fragment thereof as described herein comprises any one sequence selected from SEQ ID NO:433 and SEQ ID NO:434.

[0707] In some embodiments, a PD-1 switch molecule comprising a PD-1 polypeptide or a fragment thereof or a CD28 polypeptide or a fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence listed in Table 8 or a fragment thereof. In some embodiments, a PD-1 switch molecule comprising a PD-1 polypeptide or a fragment thereof or a CD28 polypeptide or a fragment thereof comprises any one sequence listed in Table 8 or a fragment thereof. In some embodiments, a PD-1 switch molecule comprising a PD-1 polypeptide or a fragment thereof or a CD28 polypeptide or a fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NO: 1239 and SEQ ID NO: 1244. In some embodiments, a PD-1 switch molecule comprising a PD-1 polypeptide or a fragment thereof or a CD28 polypeptide or a fragment thereof comprises any one sequence selected from SEQ ID NO: 1239 and SEQ ID NO: 1244. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding a sequence of SEQ ID NO: 1256. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a sequence encoding PD-1 N-Loop. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding a sequence of SEQ ID NO: 1257. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a sequence encoding PD-1 IgV. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding a sequence of SEQ ID NO: 1258. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a sequence encoding PD-1 Stalk. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding a sequence of SEQ ID NO: 1259. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a sequence encoding PD-1 transmembrane domain. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding a sequence of SEQ ID NO: 1239 or SEQ ID NO: 1244. In some embodiments, the nucleic acid sequence encoding the CD28 poly-peptide or a fragment thereof comprises a sequence encoding CD28 intracellular domain. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding a sequence of SEQ ID NO: 1260.

[0708] In some embodiments, an IL-15 polypeptide or a fragment thereof or an IL-15Ra subunit or a fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence listed in Table 9 or a fragment thereof. In some embodiments, an IL- 15 polypeptide or a fragment thereof or an IL-15Ra subunit or a fragment thereof comprises any one sequence listed in Table 9 or a fragment thereof. In some embodiments, an IL-15 polypeptide or a fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1242, 1245, 1246, and 1253. In some embodiments, an IL-15 polypeptide or a fragment thereof comprises any one sequence selected from SEQ ID NOs: 1242, 1245, 1246, and 1253. In some embodiments, an IL-15Ra subunit or a fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, and 1262. In some embodiments, an IL-15Ra subunit or a fragment thereof comprises any one sequence selected from SEQ ID NOs: 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, and 1262. In some embodiments, an IL-15 polypeptide or a fragment thereof and an IL-15Ra subunit or a fragment thereof are operatively linked as a fusion protein. In some embodiments, an IL- 15 polypeptide or a fragment thereof and an IL-15Ra subunit or a fragment thereof are operatively linked by a linker. In some embodiments, the linker is a P2A linker. In some embodiments, the P2A linker comprises a sequence of SEQ ID NO: 1261. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the non-cleavable linker comprises a sequence of SEQ ID NO: 1243. In some embodiments, the fusion protein comprising an IL-15 polypeptide or a fragment thereof and an IL-15Ra subunit or a fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NO: 1253. In some embodiments, the fusion protein comprising an IL-15 polypeptide or a fragment thereof and an IL-15Ra subunit or a fragment thereof comprises the sequence of SEQ ID NO: 1253.

[0709] In some embodiments, a PD-1 polypeptide or a fragment thereof is fused to a CD28 polypeptide or a fragment thereof fused to an IL-15Ra subunit or a fragment thereof as a fusion protein. In some embodiments, a PD-1 polypeptide or a fragment thereof is operatively fused to a CD28 polypeptide or a fragment thereof by a linker. In some embodiments, a CD28 polypeptide or a fragment thereof is operatively fused to an IL-15Ra subunit or a fragment thereof by a linker. In some embodiments, the fusion protein comprising a PD-1 polypeptide or a fragment thereof fused to a CD28 polypeptide or a fragment thereof fused to an IL-15Ra subunit or a fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to the sequence of SEQ ID NO: 1254 or 1262. In some embodiments, the fusion protein comprising a PD-1 polypeptide or a fragment thereof fused to a CD28 polypeptide or a fragment thereof fused to an IL-15Ra subunit or a fragment thereof comprises the sequence of SEQ ID NO: 1254 or 1262.

[0710] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence comprising a sequence having a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO: 195 or SEQ ID NO: 196, a second nucleic acid sequence comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID N0s:400-402, and a third nucleic acid sequence comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs:283, 284, 285, and 286, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1239 and 1244, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1247, 1248, 1249, 1250, and 1251, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to the sequence of SEQ ID NOs: 1253, or a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1254 and 1262.

[0711] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence comprising SEQ ID NO: 195 or SEQ ID NO: 196, a second nucleic acid sequence comprising a sequence selected from SEQ ID N0s:400-402, and a third nucleic acid sequence comprising a sequence selected from SEQ ID NOs:283, 284, 285, and 286, a sequence selected from SEQ ID NOs: 1239 and 1244, a sequence selected from SEQ ID NOs: 1247, 1248, 1249, 1250, and 1251, the sequence of SEQ ID NOs: 1253, or a sequence selected from SEQ ID NOs: 1254 and 1262.

[0712] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence comprising a sequence having a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO: 195 or SEQ ID NO: 196, a second nucleic acid sequence comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID N0s:400-402, and a third nucleic acid sequence and a fourth nucleic acid sequence independently comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs:283, 284, 285, and 286, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1239 and 1244, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1247, 1248, 1249, 1250, and 1251, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to the sequence of SEQ ID NOs: 1253, or a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1254 and 1262, wherein the third nucleic acid sequence and the fourth nucleic acid sequence are not same.

[0713] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence comprising SEQ ID NO: 195 or SEQ ID NO: 196, a second nucleic acid sequence comprising a sequence selected from SEQ ID N0s:400-402, and a third nucleic acid sequence and a fourth nucleic acid sequence independently comprising a sequence selected from SEQ ID NOs:283, 284, 285, and 286, a sequence selected from SEQ ID NOs: 1239 and 1244, a sequence selected from SEQ ID NOs: 1247, 1248, 1249, 1250, and 1251, the sequence of SEQ ID NOs: 1253, or a sequence selected from SEQ ID NOs: 1254 and 1262, wherein the third nucleic acid sequence and the fourth nucleic acid sequence are not same. [0714] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence comprising a sequence having a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO: 195 or SEQ ID NO: 196, a second nucleic acid sequence comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID N0s:400-402, a third nucleic acid sequence comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs:283, 284, 285, and 286, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1239 and 1244, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1247, 1248, 1249, 1250, and 1251, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to the sequence of SEQ ID NOs: 1253, and a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1254 and 1262, and a fourth nucleic acid sequence comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs:283, 284, 285, and 286, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1239 and 1244, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1247, 1248, 1249,

1250, and 1251, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to the sequence of SEQ ID NOs: 1253, or a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1254 and 1262, wherein the third nucleic acid sequence and the fourth nucleic acid sequence are not same.

[0715] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence comprising SEQ ID NO: 195 or SEQ ID NO: 196, a second nucleic acid sequence comprising a sequence selected from SEQ ID N0s:400-402, a third nucleic acid sequence comprising a sequence selected from SEQ ID NOs:283, 284, 285, and 286, a sequence selected from SEQ ID NOs: 1239 and 1244, a sequence selected from SEQ ID NOs: 1247, 1248, 1249, 1250, and 1251, the sequence of SEQ ID NOs: 1253, or a sequence selected from SEQ ID NOs: 1254 and 1262, and a fourth nucleic acid sequence comprising a sequence selected from SEQ ID NOs:283, 284, 285, and 286, a sequence selected from SEQ ID NOs: 1239 and 1244, a sequence selected from SEQ ID NOs: 1247, 1248, 1249, 1250, and

1251, the sequence of SEQ ID NOs: 1253, or a sequence selected from SEQ ID NOs: 1254 and 1262, wherein the third nucleic acid sequence and the fourth nucleic acid sequence are not same. [0716] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, and one or more sequences selected from a nucleic acid sequence encoding a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof as described herein, a nucleic acid sequence encoding a dominant negative TGFBR2 receptor or a fragment thereof as described herein, a nucleic acid sequence encoding a PD-1 switch molecule comprising a PD- 1 polypeptide or a fragment thereof as described herein, a nucleic acid sequence encoding a CD28 polypeptide or a fragment thereof as described herein, a nucleic acid sequence encoding a PD-1 polypeptide or a fragment thereof fused to a CD28 polypeptide or a fragment thereof fused to an IL-15Ra subunit or a fragment thereof as 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 subunit or a fragment thereof as described herein.

[0717] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence comprising a sequence having a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO: 195 or SEQ ID NO: 196, a second nucleic acid sequence comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID N0s:400-402, and one or more sequences selected from a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs:283, 284, 285, and 286, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1239 and 1244, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1247, 1248, 1249, 1250, and 1251, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to the sequence of SEQ ID NOs: 1253, and a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs: 1254 and 1262.

[0718] Disclosed herein in some embodiments, are recombinant nucleic acid molecules comprising a first nucleic acid sequence comprising SEQ ID NO: 195 or SEQ ID NO: 196, a second nucleic acid sequence comprising a sequence selected from SEQ ID N0s:400-402, and one or more sequences selected from a sequence selected from SEQ ID NOs:283, 284, 285, and 286, a sequence selected from SEQ ID NOs: 1239 and 1244, a sequence selected from SEQ ID NOs: 1247, 1248, 1249, 1250, and 1251, the sequence of SEQ ID NOs: 1253, and a sequence selected from SEQ ID NOs: 1254 and 1262.

[0719] In some embodiment, the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, and the fourth nucleic acid sequence are independently operatively linked by a linker. In some embodiments, the linker is a T2A linker. In some embodiments, the T2A linker comprises a sequence of SEQ ID NO: 1238.

[0720] In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein comprises a sequence encoding an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the amino acid sequences listed in Table 10 or a fragment thereof. In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein comprises a sequence encoding any one of the amino acid sequences listed in Table 10 or a fragment thereof. In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein comprises a sequence encoding an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the amino acid sequences selected from SEQ ID NOs: 420, 424, 426, and 435. In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein comprises a sequence encoding any one of the amino acid sequences selected from SEQ ID NOs: 420, 424, 426, and 435.

[0721] In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein further comprises a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof. In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein further comprises a sequence encoding the amino acid sequence of SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof.

[0722] In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein encodes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the amino acid sequences listed in Table 10 or a fragment thereof. In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein encodes any one of the amino acid sequences listed in Table 10 or a fragment thereof. In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein encodes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the amino acid sequences selected from SEQ ID NOs: 420, 424, 426, and 435 or a fragment thereof. In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein encodes any one of the amino acid sequences selected from SEQ ID NOs: 420, 424, 426, and 435 or a fragment thereof.

[0723] In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein further encodes an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof. In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein further encodes the amino acid sequence of SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof.

[0724] In some embodiments, the recombinant nucleic acid as described herein comprises a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof. In some embodiments, the recombinant nucleic acid as described herein further comprises a sequence encoding the amino acid sequence of SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof. In some embodiments, the recombinant nucleic acid as described herein further encodes an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof. In some embodiments, the recombinant nucleic acid as described herein further encodes the amino acid sequence of SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof.

[0725] In some embodiments, the recombinant nucleic acid as described herein comprises a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:421, a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:422, a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:387, a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:423, a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO: 1261, a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:433, a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:431, and a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272.

[0726] In some embodiments, the recombinant nucleic acid as described herein comprises, from the 5’-end to the 3’-end, a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:421 operatively linked to a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:422 operatively linked to a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:387 operatively linked to a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:423 operatively linked to a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO: 1261 operatively linked to a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:433 operatively linked to a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:431 operatively linked to a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272.

[0727] In some embodiments, the recombinant nucleic acid as described herein comprises a sequence encoding the amino acid sequence of SEQ ID NO:421, a sequence encoding the amino acid sequence of SEQ ID NO:422, a sequence encoding the amino acid sequence of SEQ ID NO:387, a sequence encoding the amino acid sequence of SEQ ID NO:423, a sequence encoding the amino acid sequence of SEQ ID NO: 1261, a sequence encoding the amino acid sequence of SEQ ID NO:433, a sequence encoding the amino acid sequence of SEQ ID NO:431, and a sequence encoding the amino acid sequence of SEQ ID NO:272.

[0728] In some embodiments, the recombinant nucleic acid as described herein comprises, from the 5’-end to the 3’-end, a sequence encoding the amino acid sequence of SEQ ID NO:421 operatively linked to a sequence encoding the amino acid sequence of SEQ ID NO:422 operatively linked to a sequence encoding the amino acid sequence of SEQ ID NO: 387 operatively linked to a sequence encoding the amino acid sequence of SEQ ID NO:423 operatively linked to a sequence encoding the amino acid sequence of SEQ ID NO: 1261 operatively linked to a sequence encoding the amino acid sequence of SEQ ID NO:433 operatively linked to a sequence encoding the amino acid sequence of SEQ ID NO:431 operatively linked to a sequence encoding the amino acid sequence of SEQ ID NO:272. [0729] In some embodiments, the recombinant nucleic acid as described herein encodes an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:421, an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:422, an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:387, an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:423, an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO: 1261, an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:433, an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:431, and an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272.

[0730] In some embodiments, the recombinant nucleic acid as described herein encodes, from the N-terminus to the C-terminus, an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:421 operatively linked to an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:422 operatively linked to an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:387 operatively linked to an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:423 operatively linked to an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO: 1261 operatively linked to an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:433 operatively linked to an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:431 operatively linked to an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272. [0731] In some embodiments, the recombinant nucleic acid as described herein encodes the amino acid sequence of SEQ ID NO:421, the amino acid sequence of SEQ ID NO:422, the amino acid sequence of SEQ ID NO:387, the amino acid sequence of SEQ ID NO:423, the amino acid sequence of SEQ ID NO: 1261, the amino acid sequence of SEQ ID NO:433, the amino acid sequence of SEQ ID NO:431, and the amino acid sequence of SEQ ID NO:272. [0732] In some embodiments, the recombinant nucleic acid as described herein encodes, from the N-terminus to the C-terminus, the amino acid sequence of SEQ ID NO:421 operatively linked to the amino acid sequence of SEQ ID NO:422 operatively linked to the amino acid sequence of SEQ ID NO:387 operatively linked to the amino acid sequence of SEQ ID NO:423 operatively linked to the amino acid sequence of SEQ ID NO: 1261 operatively linked to the amino acid sequence of SEQ ID NO:433 operatively linked to the amino acid sequence of SEQ ID NO:431 operatively linked to the amino acid sequence of SEQ ID NO:272.

[0733] In some embodiments, the recombinant nucleic acid as described herein comprises a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:421, a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:422, a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:387, a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:423, a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO: 1261, a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:437, a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:431, and a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272.

[0734] In some embodiments, the recombinant nucleic acid as described herein comprises, from the 5’-end to the 3’-end, a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:421 operatively linked to a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:422 operatively linked to a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:387 operatively linked to a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:423 operatively linked to a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO: 1261 operatively linked to a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:437 operatively linked to a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:431 operatively linked to a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272.

[0735] In some embodiments, the recombinant nucleic acid as described herein comprises a sequence encoding the amino acid sequence of SEQ ID NO:421, a sequence encoding the amino acid sequence of SEQ ID NO:422, a sequence encoding the amino acid sequence of SEQ ID NO:387, a sequence encoding the amino acid sequence of SEQ ID NO:423, a sequence encoding the amino acid sequence of SEQ ID NO: 1261, a sequence encoding the amino acid sequence of SEQ ID NO:437, a sequence encoding the amino acid sequence of SEQ ID NO:431, and a sequence encoding the amino acid sequence of SEQ ID NO:272.

[0736] In some embodiments, the recombinant nucleic acid as described herein comprises, from the 5’-end to the 3’-end, a sequence encoding the amino acid sequence of SEQ ID NO:421 operatively linked to a sequence encoding the amino acid sequence of SEQ ID NO:422 operatively linked to a sequence encoding the amino acid sequence of SEQ ID NO: 387 operatively linked to a sequence encoding the amino acid sequence of SEQ ID NO:423 operatively linked to a sequence encoding the amino acid sequence of SEQ ID NO: 1261 operatively linked to a sequence encoding the amino acid sequence of SEQ ID NO:437 operatively linked to a sequence encoding the amino acid sequence of SEQ ID NO:431 operatively linked to a sequence encoding the amino acid sequence of SEQ ID NO:272.

[0737] In some embodiments, the recombinant nucleic acid as described herein encodes an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:421, an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:422, an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:387, an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:423, an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO: 1261, an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:437, an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:431, and an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272.

[0738] In some embodiments, the recombinant nucleic acid as described herein encodes, from the N-terminus to the C-terminus, an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:421 operatively linked to an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:422 operatively linked to an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:387 operatively linked to an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:423 operatively linked to an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO: 1261 operatively linked to an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:437 operatively linked to an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:431 operatively linked to an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272.

[0739] In some embodiments, the recombinant nucleic acid as described herein encodes the amino acid sequence of SEQ ID NO:421, the amino acid sequence of SEQ ID NO:422, the amino acid sequence of SEQ ID NO:387, the amino acid sequence of SEQ ID NO:423, the amino acid sequence of SEQ ID NO: 1261, the amino acid sequence of SEQ ID NO:437, the amino acid sequence of SEQ ID NO:431, and the amino acid sequence of SEQ ID NO:272. [0740] In some embodiments, the recombinant nucleic acid as described herein encodes, from the N-terminus to the C-terminus, the amino acid sequence of SEQ ID NO:421 operatively linked to the amino acid sequence of SEQ ID NO:422 operatively linked to the amino acid sequence of SEQ ID NO:387 operatively linked to the amino acid sequence of SEQ ID NO:423 operatively linked to the amino acid sequence of SEQ ID NO: 1261 operatively linked to the amino acid sequence of SEQ ID NO:437 operatively linked to the amino acid sequence of SEQ ID NO:431 operatively linked to the amino acid sequence of SEQ ID NO:272.

[0741] 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’-0-M0E), 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. 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 [0742] Further disclosed herein, in some embodiments, are vectors comprising the recombinant nucleic acid molecules as described herein. In some embodiments, the vector comprises a nucleic acid molecule encoding a sequence encoding a TFP as described herein, a sequence encoding CXCR6 or a fragment thereof as descried herein, a sequence encoding a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof as described herein, a dominant negative TGFBR2 receptor or a fragment thereof as described herein, a PD-1 polypeptide or a fragment thereof, a PD-1 switch molecule comprising a PD-1 polypeptide or a fragment thereof as described herein, a CD28 polypeptide or a fragment thereof as described herein, a PD-1 polypeptide or a fragment thereof fused to a CD28 polypeptide or a fragment thereof fused to an IL-15Ra subunit or a fragment thereof as described herein, an IL- 15 polypeptide or a fragment thereof described herein, an IL-15Ra subunit or a fragment thereof as described herein, or a combination thereof. In one aspect, the vector can be directly transduced into a cell, e.g., a T cell. In one aspect, the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs. In one aspect, the vector is capable of expressing the TFP construct, a CXCR6 or a functional fragment thereof as described herein construct, a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof as described herein construct, a dominant negative TGFBR2 receptor or a fragment thereof as described herein construct, a PD-1 polypeptide or a fragment thereof as described herein construct, a PD-1 switch molecule comprising a PD-1 polypeptide or a fragment thereof as described herein, a CD28 polypeptide or a fragment thereof as described herein construct, a PD-1 polypeptide or a fragment thereof fused to a CD28 polypeptide or a fragment thereof fused to an IL-15Ra subunit or a fragment thereof as described herein construct, an IL-15 polypeptide or a fragment thereof described herein construct, an IL-15Ra subunit or a fragment thereof as described herein construct, or a combination thereof in mammalian T cells. In one aspect, the mammalian T cell is a human T cell.

[0743] In some embodiments, 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 embodiments, the vector is an AAV6 vector. In some embodiments, the vector further comprises a promoter. In some embodiments, the vector is an in vitro transcribed vector.

[0744] The present disclosure further provides a vector comprising a nucleic acid molecule encoding a sequence encoding a TFP as described herein, a sequence encoding CXCR6 or a fragment thereof as descried herein, a sequence encoding a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof as described herein, a sequence encoding a dominant negative TGFBR2 receptor or a fragment thereof as described herein, a PD-1 switch molecule comprising a PD-1 polypeptide or a fragment thereof as described herein, a CD28 polypeptide or a fragment thereof as described herein, a PD-1 polypeptide or a fragment thereof fused to a CD28 polypeptide or a fragment thereof fused to an IL-15Ra subunit or a fragment thereof as described herein, an IL-15 polypeptide or a fragment thereof described herein, an IL-15Ra subunit or a fragment thereof as described herein, or a combination thereof. In one aspect, a vector encoding the nucleic acid molecules as 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 CXCR6 or a fragment thereof as descried herein construct, a PD-1 switch molecule comprising a PD-1 polypeptide or a fragment thereof as described herein construct, a PD-1 polypeptide or a fragment thereof fused to a CD28 polypeptide or a fragment thereof fused to an IL-15Ra subunit or a fragment thereof as described herein construct, an IL-15 polypeptide or a fragment thereof described herein construct, an IL-15Ra subunit or a fragment thereof as described herein construct, or a combination thereof in mammalian T cells. In one aspect, the mammalian T cell is a human T cell.

[0745] 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.

[0746] 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 nonproliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0747] In another embodiment, the vector comprising the nucleic acid sequence encoding the desired TFP and the nucleic acid sequence encoding CXCR6 or a functional fragment thereof of the present disclosure is an adenoviral vector (A5/35). In another embodiment, the expression of the nucleic acid encoding TFPs as described herein and the nucleic acid encoding CXCR6 or a functional fragment thereof as described herein 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.

[0748] The nucleic acid sequence encoding the desired TFP and the nucleic acid sequence encoding CXCR6 or a functional fragment thereof of the present disclosure may be used in multi ci str onic vectors or vectors expressing several proteins in the same transcriptional unit. Such vectors may use internal ribosomal entry sites (IRES). Since IRES are not functional in all hosts and do not allow for the stoichiometric expression of multiple protein, self-cleaving peptides may be used instead. For example, several viral peptides are cleaved during translation and allow for the expression of multiple proteins form a single transcriptional unit. Such peptides include 2A-peptides, or 2A-like sequences, from members of the Picornaviridae virus family. See for example Szymczak et al., 2004, Nature Biotechnology; 22:589-594. In some embodiments, the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein encodes the TFP in frame with CXCR6 or a functional fragment thereof as described herein, with the two sequences separated by a self-cleaving peptide, such as a 2A sequence, or a T2A sequence.

[0749] 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.

[0750] 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.

[0751] 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). [0752] 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.

[0753] 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. [0754] An example of a promoter that is capable of expressing a TFP transgene as described herein and CXCR6 or a functional fragment thereof as described herein 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 and 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.

[0755] In order to assess the expression of a TFP polypeptide as described herein and CXCR6 or a functional fragment thereof as described herein, 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.

[0756] 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, betagalactosidase, 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.

[0757] 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.

[0758] 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.

[0759] 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.

[0760] 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.

[0761] 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. [0762] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 °C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0763] 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 (ELIS As and western blots) or by assays described herein to identify agents falling within the scope of the present disclosure.

[0764] The present disclosure further provides a vector comprising a nucleic acid molecule encoding a TFP as described herein and a nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein. In one aspect, a vector comprising a nucleic acid molecule encoding a TFP as described herein and a nucleic acid molecule encoding CXCR6 or a functional fragment thereof as 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 as described herein and the CXCR6 or a functional fragment thereof construct as described herein in mammalian T cells. In one aspect, the mammalian T cell is a human T cell.

[0765] 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).

[0766] The TFP-encoding nucleic acid construct as described herein with or without a nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein can be cloned into a lentiviral expression vector and expression validated based on the quantity and quality of the effector T cell response of transduced T cells in response to MSLN+ target cells. Effector T cell responses include, but are not limited to, cellular expansion, proliferation, doubling, cytokine production and target cell lysis or cytolytic activity (i.e., degranulation). [0767] In some embodiments, the vectors comprising the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein comprises the pLRPO backbone, the pLKaUS backbone, or the pLCUS backbone. In some embodiments, the vectors comprising the recombinant nucleic acid molecule encoding any one of the amino acid sequences selected from SEQ ID NOs: 420, 424, 426, and 435 or a fragment thereof comprises the pLRPO backbone, the pLKaUS backbone, or the pLCUS backbone. In some embodiments, the vectors comprising the recombinant nucleic acid molecule comprising a sequence encoding the amino acid sequence of SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof comprises the pLRPO backbone, the pLKaUS backbone, or the pLCUS backbone. In some embodiments, the vectors comprising the recombinant nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof comprises the pLRPO backbone, the pLKaUS backbone, or the pLCUS backbone.

Recombinant RNAs

[0768] Disclosed herein are methods for producing in vitro transcribed RNA encoding TFPs as described herein and in vitro transcribed RNA encoding CXCR6 or a functional fragment thereof as described herein. The present disclosure also includes a TFP as described herein encoding RNA construct and a CXCR6 or a functional fragment thereof as described herein 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 a sequence for the TFP as described herein and a sequence for CXCR6 or a functional fragment thereof as described herein as described herein.

[0769] In one aspect, the anti-TAA TFP as described herein and CXCR6 or a functional fragment thereof as described herein is encoded by messenger RNAs (mRNAs). In one aspect, the mRNA encoding the anti-TAA TFP as described herein and the mRNA encoding CXCR6 or a functional fragment thereof as described herein are introduced into a T cell for production of a T cell expressing the TFP as described herein and CXCR6 or a functional fragment thereof as described herein. In one embodiment, the in vitro transcribed RNA encoding a TFP as described herein and the in vitro transcribed RNA encoding CXCR6 or a functional fragment thereof as 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 and/or CXCR6 or a functional fragment thereof 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.

[0770] PCR can be 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.

[0771] Any DNA polymerase useful for PCR can be used in the methods as described herein. The reagents and polymerase are commercially available from a number of sources.

[0772] 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.

[0773] 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.

[0774] 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.

[0775] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5’ end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0776] 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.

[0777] On a linear DNA template, phage T7 RNA polymerase can extend the 3’ end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003).

[0778] 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. [0779] 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.

[0780] 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.

[0781] 5’ caps can also provide stability to RNA molecules. In some embodiments, RNAs produced by the methods as described 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)).

[0782] The RNAs produced by the methods as described 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.

[0783] 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 [0784] Disclosed herein, in some embodiments, are cells comprising the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein, the polypeptide as described herein, or the vectors as described herein. Disclosed herein, in some embodiments, are cells comprising the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein, the polypeptide as described herein, or the vectors as described herein, wherein cells comprise the sequence encoding a TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein. 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 aP T cell. In some embodiments, the T cell is a human y6 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 y6 T cells and do not comprise a functional disruption of an endogenous TCR. In some embodiments, the y6 T cells are V<51+ V<52- y<5 T cells. In some embodiments, the y6 T cells are V<51- V<52+ y<5 T cells. In some embodiments, the y6 T cells are V<51- V<52- y<5 T cells.

[0785] The present disclosure provides genetically-modified immune cells and populations thereof and methods for producing the same. In some embodiments, the genetically-modified immune cells of the presently disclosed compositions and methods are human immune cells. In some embodiments, the immune cells are T cells, or cells derived therefrom. In other embodiments, the immune cells are natural killer (NK) cells, or cells derived therefrom. In still other embodiments, the immune cells are B cells, or cells derived therefrom. In yet other embodiments, the immune cells are monocyte or macrophage cells or cells derived therefrom. [0786] Disclosed herein, in some embodiments, are cells comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein may have enhanced survival rate, enhanced effector function, and/or enhanced cytotoxicity compared to cells that do not comprise the sequence encoding TFP as described herein, and the sequence encoding CXCR6 or a functional fragment thereof as described herein. In some embodiments, the cell has enhanced survival rate compared to a cell that does not have the sequence encoding CXCR6 or a functional fragment thereof as described herein. In some embodiments, the cell has enhanced effector function compared to a cell that does not have the sequence encoding CXCR6 or a functional fragment thereof as described herein. In some embodiments, the cell has enhanced cytotoxicity compared to a cell that does not have the sequence encoding CXCR6 or a functional fragment thereof as described herein. [0787] Disclosed herein, in some embodiments, are cells comprising the sequence encoding TFP as described herein, and the sequence encoding CXCR6 or a functional fragment thereof as described herein may have increased longevity compared to cells that do not comprise the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein. In some embodiments, the longevity of the cell is increased compared to a cell that does not comprise a nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein.

[0788] Disclosed herein, in some embodiments, are cells comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein may have increased persistence compared to cells that do not comprise the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein. In some embodiments, the persistence of the cell is increased compared to a cell that does not comprise a nucleic acid sequence encoding CXCR6 or a functional fragment thereof.

[0789] Disclosed herein, in some embodiments, are cells comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein may have increased cytotoxicity compared to cells that do not comprise the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein. In some embodiments, the cytotoxicity of the cell is increased compared to a cell that does not comprise a nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein.

[0790] Disclosed herein, in some embodiments, are cells comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein may have increased cytokine production compared to cells that do not comprise the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein. In some embodiments, the cytokine production of the cell is increased compared to a cell that does not comprise a nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein.

[0791] In some embodiments, cells as described herein retains naive and/or central memory phenotypes. In some embodiments, cells as described herein have not differentiated into terminal effector cells.

[0792] 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 as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described 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 a nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein.

[0793] 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 as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described 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 a nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein.

[0794] 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 as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described 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 a nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein.

[0795] Disclosed herein, in some embodiments, are modified immune cells, e.g., T cells comprising the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein, or the vectors as described herein. In some embodiments, the modified T cell further 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 as described herein or a TFP encoded by the sequence of the nucleic acid as described herein and a nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein. In some embodiments, the modified T cell further 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 as described herein or a TFP encoded by the sequence of the nucleic acid as described herein and a nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein. [0796] Disclosed herein, in some embodiments, are modified T cell comprising the recombinant nucleic acid disclosed above, or the vector disclosed above; wherein the modified T cell further comprises a functional disruption of an endogenous TCR. Further disclosed herein, in some embodiments, are modified T cells comprising the sequence encoding the TFP of the nucleic acid disclosed above or a TFP encoded by the sequence of the nucleic acid disclosed above encoding the TFP as described herein and a nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein, wherein the modified T cell further comprises a functional disruption of an endogenous TCR. Also disclosed herein, are modified allogenic T cell comprising the sequence encoding the TFP disclosed above or a TFP encoded by the sequence of the nucleic acid disclosed above encoding the TFP as described herein and a nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein.

[0797] In some embodiments, 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 embodiments, 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 embodiments, 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 embodiments, the functional disruption is a disruption of a gene encoding the endogenous TCR. In some embodiments, 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 embodiments, the T cell is a CD8+ T cell, a CD4+ T cell, a naive T cell, a memory stem T cell, a central memory T cell, a double negative T cell, an effector memory T cell, an effector T cell, a ThO cell, a TcO cell, a Thl cell, a Tel cell, a Th2 cell, a Tc2 cell, a Thl7 cell, a Th22 cell, a gamma delta T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a hematopoietic stem cell, or a pluripotent stem cell. 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 CD4+CD8+ T cell. In some embodiments, the T cell is a human aP T cell. In some embodiments, the T cell is a human y6 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 y6 T cells and do not comprise a functional disruption of an endogenous TCR. In some embodiments, the y6 T cells are V<51+ V<52- y<5 T cells. In some embodiments, the y6 T cells are V<51- V<52+ y<5 T cells. In some embodiments, the y6 T cells are V51- V<52- y<5 T cells. In some embodiments, the T cell is an allogenic T cell.

[0798] In some embodiments, the modified T cells further comprise a nucleic acid sequence encoding an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain. In some embodiments, the inhibitory molecule comprises the first polypeptide comprising at least a portion of PD-1 and the second polypeptide comprising a costimulatory domain and primary signaling domain. In some embodiments, a T cell expressing the TFP as descried herein and CXCR6 or a functional fragment thereof as descried herein can inhibit tumor growth when expressed in a T cell.

[0799] In some embodiments, genetically-modified immune cells of the invention comprise an inactivated TCR alpha gene and/or an inactivated TCR beta gene. Inactivation of the TCR alpha gene and/or TCR beta gene to generate the genetically-modified cells of the present disclosure occurs in at least one or both alleles where the TCR alpha gene and/or TCR beta gene is being expressed. Accordingly, inactivation of one or both genes prevents expression of the endogenous TCR alpha chain or the endogenous TCR beta chain protein. Expression of these proteins is required for assembly of the endogenous alpha/beta TCR on the cell surface. Thus, inactivation of the TCR alpha gene and/or the TCR beta gene results in genetically-modified immune that have no detectable cell surface expression of the endogenous alpha/beta TCR. In particular embodiments, the inactivated gene is a TCR alpha constant region (TRAC) gene. In some embodiments, genetically-modified immune cells of the invention comprise an inactivated B2M gene.

[0800] In some examples, the TCR alpha gene, the TRAC gene, or the TCR beta gene is inactivated by insertion of a template nucleic acid into a cleavage site in the gene. Insertion of the template nucleic acid disrupts expression of the endogenous TCR alpha chain or TCR beta chain and, therefore, prevents assembly of an endogenous alpha/beta TCR on the T cell surface. In some examples, the template nucleic acid is inserted into the TRAC gene.

[0801] In some of those embodiments wherein the genetically-modified immune cell expresses a TFP as described herein and CXCR6 or a functional fragment thereof as described herein, such cells have no detectable cell-surface expression of an endogenous T cell receptor (e.g., an alpha/beta T cell receptor). Thus, the invention further provides a population of genetically- modified immune cells that express a TFP as described herein and CXCR6 or a functional fragment thereof as described herein and have no detectable cell-surface expression of an endogenous T cell receptor (e.g., an alpha/beta T cell receptor), and in some embodiments also express a TFP as described herein and CXCR6 or a functional fragment thereof as described herein. For example, the population can include a plurality of genetically-modified immune cells of the invention which express a TFP as described herein and CXCR6 or a functional fragment thereof as described herein, and have no cell-surface expression of an endogenous T cell receptor (i.e., are TCR-).

[0802] As used herein, “detectable cell-surface expression of an endogenous TCR” refers to the ability to detect one or more components of the TCR complex (e.g., an alpha/beta TCR complex) on the cell surface of an immune cell using standard experimental methods. Such methods can include, for example, immunostaining and/or flow cytometry specific for components of the TCR itself, such as a TCR alpha or TCR beta chain, or for components of the assembled cell-surface TCR complex, such as CD3. Methods for detecting cell-surface expression of an endogenous TCR (e.g., an alpha/beta TCR) on an immune cell include those described in the examples herein, and, for example, those described in MacLeod et al. (2017) Molecular Therapy 25(4): 949-961.

[0803] In some cases, the present disclosure provides a modified allogenic immune cell, e.g., T cell comprising the sequence encoding the TFP as described herein or a TFP encoded by the sequence of the nucleic acid as described herein encoding the TFP and the sequence encoding CXCR6 or a functional fragment thereof as described herein.

[0804] In some embodiments, migration of the cells comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein is increased in response to CXCL16. In some embodiments, migration of the cells comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein is increased in response to CXCL16 by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to the cells comprising the sequence encoding TFP as described herein but do not comprise the sequence encoding CXCR6 or a functional fragment thereof. In some embodiments, migration of the cells comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein is increased in response to CXCL16 by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to the cells comprising the sequence encoding TFP as described herein but do not comprise the sequence encoding CXCR6 or a functional fragment thereof. In some embodiments, a migration rate of the cells comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein is increased in response to CXCL16 by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to the cells comprising the sequence encoding TFP as described herein but do not comprise the sequence encoding CXCR6 or a functional fragment thereof. In some embodiments, a migration rate of the cells comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein is increased in response to CXCL16 by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to the cells comprising the sequence encoding TFP as described herein but do not comprise the sequence encoding CXCR6 or a functional fragment thereof. In some embodiments, the number of the cells comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein that migrate to a tumor site is increased in response to CXCL16 by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to the cells comprising the sequence encoding TFP as described herein but do not comprise the sequence encoding CXCR6 or a functional fragment thereof. In some embodiments, the number of the cells comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein that migrate to a tumor site is increased in response to CXCL16 by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to the cells comprising the sequence encoding TFP as described herein but do not comprise the sequence encoding CXCR6 or a functional fragment thereof.

[0805] In some embodiments, a “TFP-T cell” is a T cell that has been transduced according to the methods disclosed herein and that expresses a TFP, e.g., incorporated into the natural TCR. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a CD4+ / CD8+ T cell. In some embodiments, the TFP cell is a regulatory T cell. In some embodiments, the TFP cell is an NK cell.

[0806] The term “effector T cell” includes T helper (i.e., CD4+) cells and cytotoxic (i.e., CD8+) T cells. CD4+ effector T cells contribute to the development of several immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. CD8+ effector T cells destroy virus-infected cells and tumor cells. See Seder and Ahmed, Nature Immunol., 2003, 4:835-842, incorporated by reference in its entirety, for additional information on effector T cells. The term “regulatory T cell” includes cells that regulate immunological tolerance, for ex-ample, by suppressing effector T cells. In some aspects, the regulatory T cell has a CD4+CD25+Foxp3+ phenotype. In some aspects, the regulatory T cell has a CD8+CD25+ phenotype. See Nocentini et al., Br. J. Pharmacol., 2012, 165:2089-2099, incorporated by reference in its entirety, for additional information on regulatory T cells expressing CD70.

[0807] The term “dendritic cell” refers to a professional antigen-presenting cell capable of activating a naive T cell and stimulating growth and differentiation of a B cell.

[0808] 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.

[0809] For additional information on making and using TFP-T cells, see U.S. Patent Nos. 10,442,849, 10,358,473, 10,358,474, and 10,208,285, each of which is herein incorporated by reference.

Sources of T cells

[0810] Prior to expansion and genetic modification, a source of T cells is obtained from a subject. 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.

[0811] 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.

[0812] 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, CDl 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.

[0813] In one embodiment, a T cell population can be selected that expresses one or more of IFN-y TNF-alpha, IL- 17 A, 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. [0814] 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.

[0815] 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 lxl0⁵/mL 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. [0816] 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.

[0817] Also contemplated in the context of the present disclosure is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one aspect a blood sample or an apheresis is taken from a generally healthy subject. In certain aspects, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain aspects, the T cells may be expanded, frozen, and used at a later time. In certain aspects, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further aspect, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, and mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, and irradiation. [0818] 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 for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present 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

[0819] 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.

[0820] 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(l-2):53-63, 1999). T cells may additionally be activated and expanded in the presence of a cytokine with or without an anti-CD3 and/or CD28 antibody. Exemplary cytokines include IL-2, IL-7, IL- 15, and IL-21. In some embodiments, T cells are activated by incubation with anti-CD3/anti-CD28-conjugated beads, such as DYNABEADS® or Trans-Act® beads, for a time period sufficient for activation of the T cells. In one aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours, e.g., 24 hours. In some embodiments, T cells are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with cytokines that bind the common 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. [0821] 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 structurutally related compounds at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10, or 100 pM in the presence of feeder cells (irradiated cancer cells, PBMCs, artificial antigen presenting cells). In some embodiments, the expansion of T cells may be stimulated with synthetic phosphoantigens (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1 PP, or 2-methyl-3-butenyl-l -pyrophosphate in the presence of IL-2 for one-to-two weeks. In some embodiments, the expansion of T cells may be stimulated with immobilized anti-TCRyd (e.g., pan TCRY6) in the presence of IL-2, e.g., for approximately 14 days. In some embodiments, the expansion of T cells may be stimulated with culture of immobilized anti-CD3 antibodies (e.g., OKT3) in the presence of IL-2. In some embodiments, the aforementioned culture is maintained for about seven days prior to subculture in soluble anti-CD3, and IL-2. [0822] 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.

[0823] 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.

[0824] Once an anti-CD19, anti-BCMA, anti-CD22, anti-RORl, anti-PD-1, or anti-BAFFR, 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-BAFFR TFP are described in further detail below.

[0825] 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. [0826] In vitro expansion of T cells expressing TFT as described herein and CXCR6 or a functional fragment thereof as described herein 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-1 alpha, 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 as described herein and CXCR6 or a functional fragment thereof as described herein on day 1 using, e.g., a bicistronic lentiviral vector expressing TFP as described herein along with eGFP using a 2A ribosomal skipping sequence and CXCR6 or a functional fragment thereof as described herein. 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)).

[0827] Sustained expansion of T cells expressing TFT as described herein and CXCR6 or a functional fragment thereof as described herein in the absence of re-stimulation can also be measured (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer III particle counter following stimulation with alphaCD3/alphaCD28 coated magnetic beads on day 0, and transduction with the indicated TFP as described herein and CXCR6 or a functional fragment thereof as described herein on day 1.

[0828] Animal models can also be used to measure an activity of T cells expressing TFT as described herein and CXCR6 or a functional fragment thereof as described herein. For example, xenograft model using, e.g., human CD19-specific TFP+ T cells co-expressing CXCR6 or a functional fragment thereof as described herein 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 co-injected 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 co-expressing CXCR6 or a functional fragment thereof as described herein 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/SCZD/y-/- mice can also be analyzed. Mice are injected with leukemic cells and 3 weeks later are injected with T cells engineered to express the TFP as described herein and co-express CXCR6 or a functional fragment thereof as described herein by, e.g., a bicistronic lentiviral vector that encodes the TFP as described herein linked to eGFP and CXCR6 or a functional fragment thereof as described herein. T cells are normalized to 45-50% input GFP+ T cells co-expressing CXCR6 or a functional fragment thereof as described herein 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 groups of TFP+ T cell co-expressing CXCR6 or a functional fragment thereof as described herein are compared using the log-rank test.

[0829] Dose dependent TFP as described herein and CXCR6 or a functional fragment thereof as described herein 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 co-expressing CXCR6 or a functional fragment thereof as described herein, 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.

[0830] 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 as described herein and CXCR6 or a functional fragment thereof as described herein-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 co-expressing CXCR6 or a functional fragment thereof as described herein are identified by GFP expression using T cells that are engineered with eGFP-2A linked TFP-expressing lentiviral vectors. For TFP+ T cells co-expressing CXCR6 or a functional fragment thereof as described herein, but not expressing GFP, the TFP+ T cells co-expressing CXCR6 or a functional fragment thereof as described herein are detected with biotinylated recombinant CD 19 protein and a secondary avidin-PE conjugate. CD4+ and CD8+ expression on T cells are also simultaneously detected with specific monoclonal antibodies (BD Biosciences). Cytokine measurements are performed on supernatants collected 24 hours following re-stimulation using the human TH1/TH2 cytokine cytometric bead array kit (BD Biosciences) according the manufacturer’s instructions. Fluorescence is assessed using a FACScalibur™ flow cytometer (BD Biosciences), and data are analyzed according to the manufacturer’s instructions.

[0831] 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 NaCrCU, New England Nuclear) at 37 °C for 2 hours with frequent agitation, washed twice in complete RPMI and plated into microtiter plates. Effector T cells are mixed with target cells in the wells in complete RPMI at varying ratios of effector celktarget cell (E:T). Additional wells containing media only (spontaneous release, SR) or a 1% solution of Triton-X 100 detergent (total release, TR) are also prepared. After 4 hours of incubation at 37 °C, supernatant from each well is harvested. Released 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.

[0832] Imaging technologies can be used to evaluate specific trafficking and proliferation of TFPs in tumor-bearing animal models. Such assays have been described, e.g., in Barrett et al., Human Gene Therapy 22: 1575-1586 (2011). 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 as described herein and CXCR6 or a functional fragment thereof as described herein constructs. The T cells are stably transfected with a lentiviral construct to express firefly luciferase, and mice are imaged for bioluminescence. Alternatively, therapeutic efficacy and specificity of a single injection of TFP+ T cells co-expressing CXCR6 or a functional fragment thereof as described herein 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 as described herein and CXCR6 or a functional fragment thereof as described herein 7 days later. Animals are imaged at various time points post injection. For example, photon-density heat maps of firefly luciferase positive leukemia in representative mice at day 5 (2 days before treatment) and day 8 (24 hours post TFP+ PBLs) can be generated.

[0833] Other assays, including those described 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- BAFFR TFP constructs as described herein.

Pharmaceutical Compositions

[0834] 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 T cells of the disclosure; and (b) a pharmaceutically acceptable carrier. Disclosed herein, in some embodiments, are pharmaceutical compositions comprising: (a) the nucleic acid molecules 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.

[0835] 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.

[0836] 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. [0837] 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 as described herein may be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, in some embodiments 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).

[0838] 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. [0839] 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.

[0840] 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 and CXCR6 or a functional fragment thereof 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.

[0841] 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 embodiments larger doses of up to 40 mg per day may be used (described in U.S. Pat. No. 6,120,766).

[0842] In one embodiment, the TFP as described herein and CXCR6 or a functional fragment thereof as described herein is introduced into T cells, e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of TFP T cells co-expressing CXCR6 or a functional fragment thereof of the present disclosure, and one or more subsequent administrations of the TFP T cells co-expressing CXCR6 or a functional fragment thereof 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 coexpressing CXCR6 or a functional fragment thereof of the present disclosure are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the TFP T cells coexpressing CXCR6 or a functional fragment thereof 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 co-expressing CXCR6 or a functional fragment thereof as described herein 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 co-expressing CXCR6 or a functional fragment thereof as described herein administrations, and then one or more additional administration of the TFP T cells co-expressing CXCR6 or a functional fragment thereof as described herein (e.g., more than one administration of the TFP T cells co-expressing CXCR6 or a functional fragment thereof as described herein 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 co-expressing CXCR6 or a functional fragment thereof as described herein, 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 co-expressing CXCR6 or a functional fragment thereof as described herein are administered every other day for 3 administrations per week. In one embodiment, the TFP T cells co-expressing CXCR6 or a functional fragment thereof of the present disclosure are administered for at least two, three, four, five, six, seven, eight or more weeks.

[0843] In one aspect, TFP T cells co-expressing CXCR6 or a functional fragment thereof as described herein are generated using lentiviral viral vectors, such as lentivirus. TFP-T cells coexpressing CXCR6 or a functional fragment thereof as described herein generated that way will have stable TFP as described herein and CXCR6 or a functional fragment thereof as described herein expression.

[0844] In one aspect, TFP T cells co-expressing CXCR6 or a functional fragment thereof as described herein transiently express TFP as described herein and/or CXCR6 or a functional fragment thereof as described herein vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. Transient expression of TFPs as described herein and/or CXCR6 or a functional fragment thereof as described herein can be affected by RNA TFP as described herein and/or CXCR6 or a functional fragment thereof as described herein vector delivery. In one aspect, the TFP as described herein RNA and/or CXCR6 or a functional fragment thereof as described herein RNA is transduced into the T cell by electroporation.

[0845] A potential issue that can arise in patients being treated using T cells transiently expressing TFP as described herein and CXCR6 or a functional fragment thereof as described herein (particularly with T cells expressing murine scFv bearing TFP) is anaphylaxis after multiple treatments.

[0846] 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.

[0847] 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 T cells

[0848] Disclosed herein, in some embodiments, are methods of producing the modified T cells of the disclosure, the method comprising introducing the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein or the vectors as described herein into the cells. The recombinant nucleic acid can comprise a sequence encoding a TFP as described herein and/or a sequence encoding CXCR6 or a functional fragment thereof as described herein. In some cases, the method can further comprise (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 as described herein. In some embodiments, 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.

[0849] 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 as described herein or recombinant nucleic acid molecule as described herein, or the vectors as described herein. In some embodiments, 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.

[0850] In some embodiments, the T cell is a human T cell. In some embodiments, 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.

[0851] In some embodiments, 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 embodiments, 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 embodiments, the nuclease is a meganuclease. 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. 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.

Gene Editing Technologies

[0852] In some embodiments, the modified immune cells, e.g., T cells, as described herein are further engineered using a gene editing technique such as clustered regularly interspaced short palindromic repeats (CRISPR®, see, e.g., US Patent No. 8,697,359), transcription activator-like effector (TALE) nucleases (TALENs, see, e.g., U.S. Patent No. 9,393,257), meganucleases (endodeoxyribonucleases having large recognition sites comprising double-stranded DNA sequences of 12 to 40 base pairs), zinc finger nuclease (ZFN, see, e.g., Urnov 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).

[0853] Recent developments of technologies to permanently alter the human genome and to introduce site-specific genome modifications in disease relevant genes lay the foundation for therapeutic applications. These technologies are now commonly known as “genome editing.” [0854] 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 B2M, and/or human leukocyte antigens (HLAs), and/or programmed cell death protein 1 (PD-1), and/or other genes. [0855] 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.

[0856] 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 a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA base pair. Compact TALENs have an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley et al. (2013), Nat Commun. 4: 1762). A Compact TALEN comprises an engineered, site-specific TAL-effector DNA-binding domain fused to the nuclease domain from the I-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.

[0857] 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).

[0858] 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. [0859] 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.

[0860] 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. [0861] 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 embodiments, physical delivery methods can be selected from the methods but not limited to electroporation, microinjection, or use of ballistic particles. On the other hand, chemical delivery methods require use of complex molecules such calcium phosphate, lipid, or protein. In some embodiments, viral delivery methods are applied for gene editing techniques using viruses such as but not limited to adenovirus, lentivirus, and retrovirus.

[0862] 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: 196. The gRNA used to disrupt the TRBC locus can comprise a sequence of SEQ ID: 197.

[0863] CTCGACCAGCTTGACATCAC (SEQ ID NO: 196). [0864] ACACTGGTGTGCCTGGCCAC (SEQ ID NO: 197).

Methods of Treatment

[0865] 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 as 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 as described herein; and (b) a pharmaceutically acceptable carrier.

[0866] 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. [0867] 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 as described herein by expressing CXCR6 or a functional fragment thereof as described herein in the cell. In some embodiments, the cell is any one of cells described herein. [0868] 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 as described 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 as described herein; and (b) a pharmaceutically acceptable carrier.

[0869] In some embodiments, the modified T cell is an autologous T cell. In some embodiments, the T cell is an allogeneic T cell. In some embodiments, less cytokines are released in the subject compared a subject administered an effective amount of an unmodified control T cell. In some embodiments, less cytokines are released in the subject compared a subject administered an effective amount of a modified T cell comprising the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein, or the vector as described herein.

[0870] In some embodiments, the method comprises administering the pharmaceutical composition as described herein in combination with an agent that increases the efficacy of the pharmaceutical composition. 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.

[0871] 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 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.

[0872] In some embodiments, the cancer is adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bone cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, my elody splasti c/my el oproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, nonsmall cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, or Wilms tumor.

[0873] The present disclosure includes a type of cellular therapy where T cells are genetically modified to express a TFP as described herein and CXCR6 or a functional fragment thereof 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. 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.

[0874] 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 as described herein and CXCR6 or a functional fragment thereof 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. [0875] 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.

[0876] 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.

[0877] 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 sequence encoding a TFP as described herein and a nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein to the cells or iii) cry opreservation of the cells. In some embodiments, a nucleic acid sequence encoding a TCR gamma and/or delta constant domain is further introduced to the cells.

[0878] 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 as described 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.

[0879] 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 immune cells, e.g., 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.

[0880] 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.

[0881] 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. [0882] 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.

[0883] In some embodiments, the disease is a cancer selected from the group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, endometroid mucinous ovarian carcinoma, malignant pleural disease, pancreatic adenocarcinoma, ductal pancreatic adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, breast adenocarcinoma, a disease associated with a TAA expression, relapsed or refractory neoplastic disease, and any combination thereof. In some embodiments, the TFP targets CD70 and the disease is a cancer selected from the group consisting of T cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), an Epstein-Barr virus (EBV) + cancer, a human papilloma virus (HPV) + cancer, kidney cancer, renal cell carcinoma, lung cancer, pancreatic cancer, ovarian cancer, esophageal cancer, nasopharyngeal carcinoma, mesothelioma, glioblastoma, thymic carcinoma, breast cancer, head and neck cancer, gastric cancer, and any combinations thereof. In some embodiments, the TFP targets mesothelin (MSLN) and the disease is a cancer selected from mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, thyroid cancer, bladder cancer, ureter cancer, kidney cancer, endometrial cancer, esophageal cancer, gastric cancer, thymic carcinoma, cholangiocarcinoma, stomach cancer, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, endometroid mucinous ovarian carcinoma, pancreatic adenocarcinoma, ductal pancreatic adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, breast adenocarcinoma, and any combinations thereof. In some embodiments, the TFP targets CD 19 and the disease is a cancer selected from the group consisting of B-cell acute lymphoid leukemia (“BALL”), T-cell acute lymphoid leukemia (“TALL”), acute lymphoid 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- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin’s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, “preleukemia” which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and any combinations thereof. In some embodiments, the TFP targets MUC16 and the disease is a cancer selected from the group consisting of pancreatic cancer, ovarian cancer, stomach cancer, lung cancer, endometrial cancer, atypical cancers expressing MUC16, or any combinations thereof. In some embodiments, the TFP targets BCMA and the disease is a cancer selected from the group consisting of B-cell acute lymphoid leukemia (“BALL”), T-cell acute lymphoid leukemia (“TALL”), acute lymphoid 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- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, nonHodgkin’s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, preleukemia, and any combinations thereof. In some embodiments, the TFP targets CD79B and the disease is a cancer selected from the group consisting of lymphoma, myeloma, non-Hodgkin lymphoma (NHL), diffuse large-cell B-cell lymphoma, aggressive NHL, asymptomatic NHL, follicular lymphoma, recurrent aggressive NHL, recurrent non-chronic non-treatable NHL, non-treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (RE), acute lymphocytic leukemia (ALL), lymphoma of the head cortex brain, and any combinations thereof. In some embodiments, the TFP targets HER2 and the disease is a cancer selected from breast cancer, ovarian cancer, endometrial cancer, gastric cancer, pancreatic cancer, prostate cancer, salivary gland cancer, and any combinations thereof. In some embodiments, the TFP targets PSMA and the disease is a cancer selected from the group consisting of prostate cancer, endometrial cancer, breast cancer, kidney cancer, colon cancer, and any combinations thereof. In some embodiments, the TFP targets CD20 and the disease is a cancer selected from the group consisting of non-Hodgkin lymphomas, Hodgkin's disease, acute lymphoblastic leukemias, myelomas, chronic lymphocytic leukemias, myeloblastic leukemias, and any combinations thereof.

[0884] Suitable doses of the modified T cells described herein for a therapeutic effect would be at least 10⁵ or between about 10⁵ and about IO¹⁰ cells per dose, for example, preferably in a series of dosing cycles. An exemplary dosing regimen consists of four one-week dosing cycles of escalating doses, starting at least at about 10⁵ cells on Day 0, for example increasing incrementally up to a target dose of about IO¹⁰ cells within several weeks of initiating an intrapatient dose escalation scheme. Suitable modes of administration include intravenous, subcutaneous, intracavitary (for example by reservoir-access device), intraperitoneal, and direct injection into a tumor mass.

[0885] An effective amount or sufficient number of the modified T cells is present in the composition and introduced into the subject such that long-term, specific, anti-cancer and/or anti-tumor responses are established to reduce the size of a tumor or eliminate tumor growth or regrowth than would otherwise result in the absence of such treatment. Desirably, the amount of T cells introduced into the subject causes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in tumor size when compared to otherwise same conditions wherein the T cells are not present.

[0886] Accordingly, the amount of T cells administered should take into account the route of administration and should be such that a sufficient number of the T cells will be introduced so as to achieve the desired therapeutic response. Furthermore, the amounts of each active agent included in the compositions described herein (e.g., the amount per each cell to be contacted or the amount per certain body weight) can vary in different applications.

[0887] Disclosed herein, in some embodiments, is a method of increasing an activity of a cell expressing a recombinant nucleic acid comprising a sequence encoding a TFP as described herein comprising expressing in the cell. In some embodiments, the activity of a cell expressing a recombinant nucleic acid comprising a sequence encoding a TFP as described herein increases by expressing CXCR6 or a functional fragment thereof as described herein in the cell by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to the cells comprising the sequence encoding TFP as described herein, but do not express CXCR6 or a functional fragment thereof. In some embodiments, the activity of a cell expressing a recombinant nucleic acid comprising a sequence encoding a TFP as described herein increases by expressing CXCR6 or a functional fragment thereof as described herein in the cell by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to the cells comprising the sequence encoding TFP as described herein, but do not express CXCR6 or a functional fragment thereof.

[0888] In some embodiments, the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein has enhanced migration compared to migration of a cell that comprises the sequence encoding the TFP as described herein and does not express CXCR6 or functional fragment thereof. In some embodiments, the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein has enhanced migration by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to migration of a cell that comprises the sequence encoding the TFP as described herein and does not express the CXCR6 or functional fragment thereof. In some embodiments, the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein has enhanced migration by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to migration of a cell that comprises the sequence encoding the TFP as described herein and does not express the CXCR6 or functional fragment thereof.

[0889] In some embodiments, the migration rate of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein in response to CXCL16 is faster compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, the migration rate of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein in response to CXCL16 is faster by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, the migration rate of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein in response to CXCL16 is faster by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof.

[0890] In some embodiments, more number of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein migrates in response to CXCL16 compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, more number of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein migrates in response to CXCL16 by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, more number of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein migrates in response to CXCL16 by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. [0891] In some embodiments, the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein has enhanced tumor lysis activity compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein has enhanced tumor lysis activity by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein has enhanced tumor lysis activity by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. [0892] In some embodiments, the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein has increased cytokine production compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein has increased cytokine production by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein has increased cytokine production by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof.

[0893] Disclosed herein, in some embodiments, is a method of enhancing migration of a cell expressing a recombinant nucleic acid comprising a sequence encoding TFP as described herein comprising expressing CXCR6 or a functional fragment thereof as described herein in the cell. In some embodiments, migration of a cell expressing a recombinant nucleic acid comprising a sequence encoding TFP as described herein and expressing CXCR6 or a functional fragment thereof as described herein in the cell is enhanced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, migration of a cell expressing a recombinant nucleic acid comprising a sequence encoding TFP as described herein and expressing CXCR6 or a functional fragment thereof as described herein in the cell is enhanced by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, the migration rate of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein in response to CXCL16 is faster compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, the migration rate of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein in response to CXCL16 is faster by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, the migration rate of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein in response to CXCL16 is faster by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, more number of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein migrates in response to CXCL16 compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, more number of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein migrates in response to CXCL16 by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, more number of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein migrates in response to CXCL16 by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof.

[0894] Disclosed herein, in some embodiments, is a method of enhancing tumor lysis activity of a cell expressing a recombinant nucleic acid comprising a sequence encoding a TFP as described herein comprising expressing CXCR6 or a functional fragment thereof as described herein in the cell. In some embodiments, the tumor lysis activity of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein is enhanced as compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, the tumor lysis activity of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein is enhanced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, the tumor lysis activity of the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein is enhanced by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof.

[0895] Disclosed herein, in some embodiments, is a method of increasing cytokine production by a cell expressing a recombinant nucleic acid comprising a sequence encoding a TFP as described herein comprising expressing CXCR6 or a functional fragment thereof as described herein in the cell. In some embodiments, the cytokine production by the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein is increased compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, the cytokine production by the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. In some embodiments, the cytokine production by the cell comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional 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 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof.

[0896] Disclosed herein, in some embodiments, are cells comprising the sequence encoding TFP as described herein and the sequence encoding CXCR6 or a functional fragment thereof as described herein may have enhanced survival rate, enhanced effector function, and/or enhanced cytotoxicity compared to cells that do not comprise the sequence encoding TFP as described herein, and the sequence encoding CXCR6 or a functional fragment thereof as described herein. In some embodiments, the cell has enhanced survival rate compared to a cell that does not have CXCR6 or a functional fragment thereof as described herein. In some embodiments, the cell has enhanced survival rate compared to a cell that does not have CXCR6 or a functional fragment thereof as described herein. In some embodiments, the cell has enhanced survival rate by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to a cell that does not have CXCR6 or a functional fragment thereof as described herein. In some embodiments, the cell has enhanced survival rate by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to a cell that does not have CXCR6 or a functional fragment thereof as described herein.

[0897] In some embodiments, the cell has enhanced cytotoxicity compared to a cell that does not have CXCR6 or a functional fragment thereof as described herein. In some embodiments, the cell has enhanced cytotoxicity by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, 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 to a cell that does not have CXCR6 or a functional fragment thereof as described herein. In some embodiments, the cell has enhanced cytotoxicity by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 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 to a cell that does not have CXCR6 or a functional fragment thereof as described herein.

Combination Therapies

[0898] A modified immune cells, e.g., 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.

[0899] In some embodiments, the “at least one additional therapeutic agent” includes a modified immune cell, e.g., T cell. Also provided are T cells that express multiple TFPs as described herein, which bind to the same or different target antigens, or same or different epitopes on the same target antigen, and multiple CXCR6 polypeptides or functional fragments thereof as described herein. Also provided are populations of T cells in which a first subset of T cells express a first TFP and a first CXCR6 or a functional fragment thereof as described herein, and a second subset of T cells express a second TFP and a second CXCR6 or a functional fragment thereof as described herein.

[0900] A modified immune cell, e.g., 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 immune cell, e.g., T cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.

[0901] In some embodiments, the modified immune cell, e.g., T cell, as described herein provided herein are administered with at least one additional therapeutic agent. Any suitable additional therapeutic agent may be administered with a modified immune cell, e.g., T cell, as described herein provided herein. In some aspects, the additional therapeutic agent is selected from radiation, a cytotoxic agent, a chemotherapeutic agent, a cytostatic agent, an anti-hormonal agent, an EGFR inhibitor, an immunostimulatory agent, an anti-angiogenic agent, and combinations thereof.

[0902] In further aspects, a modified immune cell, e.g., T cell, as 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, irradiation, and peptide vaccine, such as that described in Izumoto et al., 2008 J. Neurosurg. 108:963-971.

[0903] In one embodiment, the subject can be administered an agent which reduces or ameliorates a side effect associated with the administration of a modified immune cell, e.g., 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 as described 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).

[0904] In one embodiment, the subject can be administered an agent which enhances the activity of a modified immune cell, e.g., 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 as described herein and CXCR6 or a functional fragment thereof as described herein-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 as described herein and/or the nucleic acid that encodes CXCR6 or a functional fragment thereof as described herein. 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 T cell immunoglobulin and mucin-domain containing-3 (TIM3). In an embodiment, the agent is an antibody or antibody fragment that binds to Lymphocyte-activation gene 3 (LAG).

[0905] 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 expresses the TFP as described herein and CXCR6 or a functional fragment thereof as described herein. In another embodiment, the fusion protein is expressed by a cell, e.g., a T cell that does not express an anti-TAA TFP as described herein and CXCR6 or a functional fragment thereof as described herein.

EXAMPLES

[0906] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples specifically point out various aspects of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0907] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided herein.

[0908] The entire disclosures of all patent and non-patent publications cited herein are each incorporated by reference in their entireties for all purposes. Background for Examples

[0909] T-Cell Receptor (TCR) is formed by a complex of dimers TCRa/p, CD3y/s, CD36/s and the homodimer CD3< In some particular T cells, TCRy/5 are expressed instead of TCRa/p to form a functional TCR. TCRa/p/y/6 have a constant domain common to all T-cells and a variable domain specific to an antigen. TRAC, TRBC, TRGC and TRDC genes encode for the constant C-terminal region of TCRa, TCRP, TCRy and TCR5 respectively. Despite high structural homology between those molecules, TCRa only pairs with TCRP and TCRy only pairs with TCR5. Hence, a TCR complex is formed with TCR /p in /p T cells or with TCRy/5 in y/6 T cells.

Example 1:

[0910] RNA-seq data for CXCL16 expression for a number of tumor samples from 33 different tumor types was obtained and analyzed from the TCGA database. FIG. 1 shows the gene expression profile of CXCL16 across the 33 different tumor types (black dot indicates the expression in each sample and black line for each cancer type shows mean expression). The results presented herein demonstrate that tumor cells from a variety of different tumors show high CXCL16 expression levels. In FIG. 1, the tumor types are abbreviated as follows:

ACC: Adrenocortical carcinoma

BLCA: Bladder Urothelial Carcinoma

BRCA: Breast invasive carcinoma

CESC: Cervical squamous cell carcinoma and endocervical adenocarcinoma

CHOL: Cholangio carcinoma

COAD: Colon adenocarcinoma

DLBC: Lymphoid Neoplasm Diffuse Large B-cell Lymphoma

ESCA: Esophageal carcinoma

GBM: Glioblastoma multiforme

HNSC: Head and Neck squamous cell carcinoma

KICH: Kidney Chromophobe

KIRC: Kidney renal clear cell carcinoma

KIRP: Kidney renal papillary cell carcinoma

LAML: Acute Myeloid Leukemia LGG: Brain Lower Grade Glioma LIHC: Liver hepatocellular carcinoma LUAD: Lung adenocarcinoma

LUSC: Lung squamous cell carcinoma

MESO: Mesothelioma

OV: Ovarian serous cystadenocarcinoma

PAAD: Pancreatic adenocarcinoma

PCPG: Pheochromocytoma and Paraganglioma

PRAD: Prostate adenocarcinoma

READ: Rectum adenocarcinoma

SARC: Sarcoma

SKCM: Skin Cutaneous Melanoma

STAD: Stomach adenocarcinoma

TGCT: Testicular Germ Cell Tumors

THCA: Thyroid carcinoma THYM: Thymoma

UCEC: Uterine Corpus Endometrial Carcinoma

UCS: Uterine Carcinosarcoma

UVM: Uveal Melanoma

Example 2: Generation of T cell receptor fusion protein T Cells

[0911] TFP constructs are generated as previously described. An anti-MSLN binder is 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 a lentiviral vector (e.g., pLRPO, pLRPC, pLCUS, or pLKaUS). In some embodiments, the TFP used is TC-210 (an anti-MSLN MHle VHH antibody linked to CD3 epsilon) having the sequence of SEQ ID NO: 195.

Source of TCR Subunits

[0912] 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 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.

[0913] TCR beta chain constant region (Homo sapiens):

VEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVST DPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP VTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKR KDF (SEQ ID NO: 16).

[0914] The murine TCR beta chain constant region canonical sequence (murine TCR beta chain constant region 1 canonical sequence) is:

EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVST DPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQN ISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 152).

[0915] The murine TCR alpha chain constant (mTRAC) region canonical sequence is: XIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKS NGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRI LLLKVAGFNLLMTLRLWSS (SEQ ID NO: 146).

[0916] TCR beta chain (Homo sapiens):

PVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVSWYQQSLDQGLQFLIQYYNGEERAK GNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSPRTGLNTEAFFGQGTRLTVVEDL NKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQP LKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI VSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 19).

[0917] TCR delta constant region version 1 (Homo sapiens):

SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKL GKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVH TEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFF (SEQ ID NO:20).

[0918] human TCR gamma chain constant region canonical sequence (TCR gamma constant region (Homo sapiens) (or [hs]TRGC(l-173))):

DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN TMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN CSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS (SEQ ID NO:21).

[0919] TCR delta constant region version 2 (Homo sapiens):

SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKL GKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVH TEKVNMMSLTVLGLRMLFAKTVAVNFLLTAK (SEQ ID NO:22).

[0920] The human CD3-epsilon polypeptide canonical sequence is:

MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEIL WQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRA RVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQR GQNKERPPPVPNPDYEPIRKGQRDL YSGLNQRRI (SEQ ID NO: 124).

[0921] The mature human CD3-epsilon polypeptide sequence is:

DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHL SLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICIT GGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDL YSGLNQRRI (SEQ ID NO:258).

[0922] The signal peptide of human CD3s is:

MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO: 125).

[0923] The extracellular domain of human CD3s is:

DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHL SLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 126). [0924] The transmembrane domain of human CD3s is:

VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID NO: 127).

[0925] The intracellular domain of human CD3s is:

KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDL YSGLNQRRI (SEQ ID NO: 128).

[0926] The human CD3-gamma polypeptide canonical sequence is:

MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKD GKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATI SGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHL QGNQLRRN (SEQ ID NO: 129).

[0927] The mature human CD3-gamma polypeptide sequence is:

QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAK DPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAG QDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 130). [0928] The signal peptide of human CD3y is:

MEQGKGLAVLILAIILLQGTLA (SEQ ID NO: 131).

[0929] The extracellular domain of human CD3y is:

QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAK DPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS (SEQ ID NO: 132).

[0930] The transmembrane domain of human CD3 y is: GFLFAEIVSIFVLAVGVYFIA (SEQ ID NO: 133).

[0931] The intracellular domain of human CD3y is:

GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 134). [0932] The human CD3-delta polypeptide canonical sequence is:

MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLG KRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALG VFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKS (SEQ ID

NO:135).

[0933] The mature human CD3-delta polypeptide sequence is:

FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDK

ESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQA LLRNDQVYQPLRDRDDAQYSHLGGNWARNKS (SEQ ID NO: 136).

[0934] The signal peptide of human CD36 is:

MEHSTFLSGLVLATLLSQVSP (SEQ ID NO: 137).

[0935] The extracellular domain of human CD36 is:

FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDK ESTVQVHYRMCQSCVELDPATVA (SEQ ID NO: 138).

[0936] The transmembrane domain of human CD36 is: GIIVTDVIATLLLALGVFCFA (SEQ ID NO: 139).

[0937] The intracellular domain of human CD36 is:

GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO: 140).

[0938] The human CD3-zeta polypeptide canonical sequence is:

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:141).

[0939] The human TCR alpha chain constant region canonical sequence is:

IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSN SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFR ILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 142).

[0940] The human TCR alpha chain human IgC sequence is:

IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSN SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS (SEQ ID NO: 143).

[0941] The transmembrane domain of the human TCR alpha chain is: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 144).

[0942] The intracellular domain of the human TCR alpha chain is:

SS (SEQ ID NO: 145).

[0943] The transmembrane domain of the murine TCR alpha chain is:

MGLRILLLKVAGFNLLMTLRLW (SEQ ID NO: 147).

[0944] The intracellular domain of the murine TCR alpha chain is: SS.

[0945] The human TCR beta chain constant region 1 (hTRBCl) canonical sequence is:

EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DF (SEQ ID NO: 148).

[0946] The human TCR beta chain 1 human IgC sequence is:

EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYE (SEQ ID NO: 149).

[0947] The transmembrane domain of the human TCR beta chain 1 is:

ILLGKATLYAVLVSALVLMAM (SEQ ID NO: 150).

[0948] The intracellular domain of the human TCR beta chain 1 is:

VKRKDF (SEQ ID NO: 151).

[0949] The human TCR beta chain 2 constant region (hTRBC2) canonical sequence is:

DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG (SEQ ID NO:371).

[0950] The transmembrane domain of the murine TCR beta chain 1 is:

ILYEILLGKATLYAVLVS TLVVMAMVK (SEQ ID NO: 153).

[0951] The intracellular domain of the murine TCR beta chain 1 is:

KRKNS (SEQ ID NO: 154).

[0952] The murine TCR beta chain constant 2 region canonical sequence is:

XDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVST DPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQN ISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO:282).

[0953] The human TCR gamma human IgC sequence is:

DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN TMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN CSKDANDTLLLQLTNTSA (SEQ ID NO: 155).

[0954] The transmembrane domain of the human TCR gamma chain is: YYMYLLLLLKSVVYFAIITCCLL (SEQ ID NO: 156).

[0955] The intracellular domain of the human TCR gamma chain is:

RRTAFCCNGEKS (SEQ ID NO: 157).

[0956] The human TCR delta chain C region canonical sequence is:

SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKL

GKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVH TEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL (SEQ ID NO:243).

[0957] The human TCR delta human IgC sequence is:

SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKL

GKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVH TEKVNMMSLTV (SEQ ID NO:265).

[0958] The transmembrane domain of the human TCR delta chain is:

LGLRMLFAKTVAVNFLLTAKLFF (SEQ ID NO: 158).

[0959] The intracellular domain of the human TCR delta chain is: L.

[0960] The human TCR alpha chain canonical sequence is:

MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLD SPIWFSAGNGSALDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHS RSTQPMHLSGEASTARTCPQEPLRGTPGGALWLGVLRLLLFKLLLFDLLLTCSCLCDPA GPLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVWG EGSYLSSYPTCPAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGAA (SEQ ID NO:573). [0961] The murine TCR alpha chain (2-137) sequence is:

IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSN GAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILL LKVAGFNLLMTLRLWSS (SEQ ID NO:207).

[0962] The murine TCR beta chain (2-173) sequence is:

DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTD PQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNIS AEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO:209).

[0963] The TCR52cl5 sequence is:

MQRIS SLIHLSLFWAGVMS A IELVPEHQT VP VSIGVP ATLRC SMKGEAIGNYYINWYRK TQGNTMTFIYREKDIYGPGFKDNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDALKR TDTDKLIFGKGTRVTVEPRSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKIT EFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKET ENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL (SEQ ID NO:256).

[0964] The TCRy9G115 sequence is:

AGHLEQPQISSTKTLSKTARLECVVSGITISATSVYWYRERPGEVIQFLVSISYDGTVRKE SGIPSGKFEVDRIPETSTSTLTIHNVEKQDIATYYCALWEAQQELGKKIKVFGPGTKLIIT DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWEEKKSNTILGSQEGN TMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN CSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS (SEQ ID NO:255).

CXCR6

[0965] In some embodiments, TFP constructs are comprised in a vector that further contains a sequence encoding CXCR6. The CXCR6 sequence may be codon optimized. In some embodiments, CXCR6 has the amino acid sequence of SEQ ID NO:400. In some embodiments, CXCR6 has the nucleotide sequence of SEQ ID NO:427. CXCR6 may be encoded in the same open reading frame and separated by a self-cleaving peptide (e.g., a P2AW or a T2A selfcleaving peptide). In some embodiments, CXCR6 is upstream from the TFP. In some embodiments, CXCR6 is downstream from the TFP. [0966] The constructs used in this study are shown in Table 1 below:

Table 1: Constructs

TFP Expression Vectors

[0967] 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).

[0968] The TFP-encoding nucleic acid constructs shown above were cloned into a lentiviral expression vector.

T-cell activation, transduction, and expansion

[0969] T cells were purified from healthy donor leukopak or PBMCs via positive selection of CD4+ and CD8+ T cells with CD4 and CD8 microbeads from Miltenyi Biotech. On day 0, T cells, freshly isolated or thawed from previously prepared frozen vials, were activated by MACS GMP T cell TransAct (Miltenyi Biotech), in the presence of human IL-7 and IL-15 (both from Miltenyi Biotech, premium grade). On day 1, activated T cells were transduced with lentivirus encoding the TFP with or without CXCR6. On day 4, the cells were washed, subcultured in fresh medium with cytokines and then expanded up to day 10 by supplementing fresh medium every 2-3 days. At each day of subculture, fresh medium with cytokines were added to maintain the cell suspension at IxlO⁶ cells/mL.

[0970] Verification of TFP expression and phenotyping of TFP -expressing cells by cell staining [0971] Following lentiviral transduction, expression of TFPs by T cells transduced with TC-210 or CXCR6-MH1 was confirmed by flow cytometry, using an anti-VHH antibody, on day 10 of cell expansion. As is shown in FIG. 2, binding of the anti-VHH antibody was detected in TFP- transduced T cells, but not in non-transduced control T cells from the same donors. TFP transduced cells with and without CXCR6 showed at least 75% transduction efficiency. Surface and intracellular expression of CXCR6 was also measured with the K041E5 antibody. As is shown in FIG. 2, little CXCR6 expression was detected in non-transduced cells or TC-210 transduced cells, whereas high levels of intracellular and surface expression of CXCR6 were detected on cells transduced with CXCR6-MH1.

[0972] The CD4:CD8 ratio of transduced cells was also determined by flow cytometry. As is shown in FIG. 3, there was no significant difference in CD4:CD8 ratio between the different TFP constructs (with or without CXCR6). The memory status of the T cells was determined by flow cytometry to detect cell surface levels of CD45RA and CCR7 as is shown in FIG. 3. There was also no significant difference in memory status between the different TFP constructs (with or without CXCR6).

Example 3: Transwell migration assay

[0973] In order to test the ability of CXCR6-expressing TFP T-cells to migrate towards a CXCL16 chemokine gradient, transwell migration assays were done in which TFP-T cells were incubated in the upper chamber of a transwell plate with the lower chamber containing recombinant CXCL16 or supernatants of tumor cells. The upper and lower chamber are separated by a porous membrane. Migration towards the CXCL16 chemokine gradient is determined by the number of cells that migrate into the lower chamber of the transwell plate. A schematic illustrating the assay is shown in FIG. 4.

[0974] To test migration of TFP T cells towards recombinant CXCL16, TFP T cells with or without CXCR6 (TC-210 or CXCR6-MH1) were placed in the upper well of the transwell plate, and recombinant CXCL16 was placed in the lower well at concentrations of 0, 6.25, 12.5, 25, or 50 ng/ml. Plates were incubated at 37°C for 4 or 15 hours and after incubation, the number of total CD3+ T cells and VHH+ T cells in the bottom well of the plate were measured. The results are shown in FIG. 5. The total number of CD3+ T cells as well as the proportion of VHH+ (TFP+) T cells at each time point and at each concentration of recombinant CXCL16 is shown. At each time point, expression of CXCR6, resulted in increased total cells in the bottom well of the transwell plate, and an increased proportion of the cells that migrated being TFP+. This indicates that expression of CXCR6 increased migration of TFP T cells towards a CXCL16 cytokine gradient.

[0975] The experiment was repeated including cells expressing TC-210, CXCR6-MH1, and MH1-WCXCR6, so that constructs expressing CXCR6 upstream and downstream of MHle could be compared. 2e5 TFP expressing T cells were placed in the upper well of the transwell plate and recombinant CXCL16 was placed in the lower well at concentrations of 0, 3.125, 6.25, 12.5, 25, 50 or 100 ng/ml. Plates were incubated at 37C for 4 hours and after incubation, the number of total CD3+ T cells and VHH+ T cells in the bottom well of the plate were measured. The results are shown in FIG. 6. The total number of CD3+ T cells as well as the proportion of VHH+ (TFP+) T cells at each concentration of recombinant CXCL16 is shown. For both the CXCR6-MH1 and MH1-WCXCR6 constructs, expression of CXCR6 resulted in increased total cells in the bottom well of the transwell plate, and an increased proportion of the cells that migrated being TFP+. This indicates that expression of CXCR6 increased migration of TFP T cells towards a CXCL16 cytokine gradient, regardless of the orientation of CXCR6 in the lentiviral vector.

[0976] To test migration of TFP T cells towards tumor cell supernatant, tumor cell lines MSTO- msln, Panc-1, Suit-2, and Suit-2-msln were cultured and the supernatant was harvested after 48 hours. Levels of soluble CXCL16 produced by each cell line is shown in FIG. 7. TFP T cells with or without CXCR6 (TC-210 or CXCR6-MH1) were placed in the upper well of the transwell plate, and the tumor cell supernatant was placed in the lower well. Plates were incubated at 37C for 4 hours and after incubation, the number of total CD3+ T cells and VHH+ T cells in the bottom well of the plate were measured. The results are shown in FIG. 7. The total number of CD3+ T cells as well as the proportion of VHH+ (TFP+) T cells is shown. For Panc- 1, Suit-2, and Suit-2-msln cell lines, expression of CXCR6, resulted in increased total cells in the bottom well of the transwell plate. For all cell lines, expression of CXCR6 resulted in an increased proportion of the cells that migrated being TFP+. This indicates that expression of CXCR6 increased migration of TFP T cells towards a CXCL16 cytokine gradient as generated by tumor cell lines.

Example 4: Luciferase-based cytotoxicity assay

[0977] The luciferase-based cytotoxicity assay assesses the cytotoxicity of T cells by indirectly measuring the luciferase enzymatic activity in the residual live target cells after co-culture. Tumor cell lines MSTO-msln, OVCAR3, Suit-2, and Panc-1 were modified to overexpress firefly luciferase via transduction with firefly luciferase encoding lentivirus followed with antibiotic selection to generate stable cell lines.

[0978] The target cells were plated at 10000 cells per well in a flat-bottom 96-well plate. T cells transduced with TC-210, CXCR6-MH1, or MH1-WCXCR6 or non-transduced T cells were added to the target cells at a 9: 1, 3 : 1, or 1 : 1 ratio. The mixture of cells was then cultured for 24 hours at 37°C with 5 % CO2 before the luciferase enzymatic activity in the live target cells was measured by the Bright-Glo® Luciferase Assay System (Promega®, Catalogue number E2610). The cells were spun into a pellet and resuspended in medium containing the luciferase substrate. The percentage of tumor cell killing was then calculated with the following formula: % Cytotoxicity = 100% x [1 - RLU (Tumor cells + T cells) / RLU (Tumor cells)]. [0979] As is shown in FIG. 8, T cells transduced with MSLN.TFP alone or combination with CXCR6 in any configuration demonstrated enhanced cytotoxicity towards target cells relative to non-transduced control T cells and the cytotoxicity was similar between TC-210, CXCR6-MH1, and MH1-WCXCR6 transduced T cells.

Example 5: Cytokine Secretion measurement by MSP

[0980] A measure of effector T-cell activation and proliferation associated with the recognition of cells bearing cognate antigen is the production of effector cytokines such as interferongamma (IFN-y), interleukin 2 (IL-2), GM-CSF and tumor necrosis factor alpha (TNF-a).

[0981] Target-specific cytokine production including IFN-y, IL-2, TNF-a and GM-CSF by T cells transduced with the vector encoding TC-210, CXCR6-MH1, or MH1-WCXCR6 or nontransduced T cells was measured from supernatants harvested 24 hours after the co-culture of T cells with target cells described in Example 4 using the U-PLEX® Biomarker Group I (hu) Assays (Meso Scale Diagnostics®, LLC, catalog number: K15067L-4).

[0982] As is shown in FIGs. 9A-9D, increased levels of IFN-y, IL-2, TNF-a, and GM-CSF were produced by T cells transduced with TC-210 when co-cultured with target cells, and T- cells transduced with MH1-WCXCR6 showed similarly elevated levels of cytokine production. T-cells transduced with CXCR6-MH1 did not show the same degree of elevation in cytokine expression levels, suggesting the orientation of CXCR6 in the lentiviral vector may affect the efficacy of the TFP and CXCR6-expressing T cells.

Example 6: Migration and cytotoxicity

[0983] Additional studies were conducted to further test activity of the CXCR6 expressing TFP T cells. The constructs used in these studies are shown below in Table 2. In addition to the CXCR6 containing constructs tested above in Examples 2-5 (i.e., CXCR6-MH1 and pLRPO- MH1-CXCR6 in Table 2), additional constructs pLCuS-MHl-CXCR6 (MH1-CXCR6 orientation, with T2AW linker on pLCuS backbone) and MH1-FCXCR6 (MH1-CXCR6 orientation and having a linker with a furin cleavage) were tested.

Table 2. Constructs tested in the studies of this Example

[0984] After transduction, expression of TFPs by T cells transduced with TC-210 or the indicated CXCR6 and MH1 TFP was confirmed by flow cytometry, using an anti-VHH antibody. Cells were also stained with CD3 and CXCR6. As shown in FIG. 10, VHH+ cells were positive for CXCR6 in cells transduced with the constructs containing CXCR6, but cells in the TC-210 group did not highly express CXCR6.

[0985] For the migration assay, a range of concentrations of CXCL16 (100, 50, 25, 12.5, 6.25, 3.125, and 0 ng/mL) was added to the lower well of a transwell plate, and 2 x 10⁵ TFP T cells (normalized to -40% VHH+) were placed in the upper well above the microporous membrane. The plate was rested at 37°C for 4 hours, then the insert was removed and migrated cells were stained for CD3 and VHH. The results are shown in FIG. 11. The total number of CD3+ T cells, total number of VHH+ cells, and the %VHH+ cells that migrated are shown. CXCR6 expressing TFP T cells migrated in response to the CXCL16 gradient. To confirm the migration was due to the CXCR6-CXCL16 interaction, a further study was conducted to incorporate an anti-CXCL16 antibody. The lower well of the transwell plate was plated with 25 or 50 ng/mL recombinant CXCL16, with or without 4pg/mL anti-CXCL16. 2 x 10⁵ TFP T cells (normalized to -40% VHH+) were added to the upper inset of the transwell plate. The plate was rested at 37°C for 4 hours, then the insert was removed and the migrated cells were stained. The results are shown in FIG. 12. The presence of the blocking anti-CXCL16 antibody limited migration of the TFP T cells.

[0986] An assay to determine both migration and cytotoxicity of migrated TFP T cells against CXCL16 tumor cell lines was conducted. 5 x 10³ tumor cells were plated in the bottom well of transwell plates, and incubated for 48 hours. MSTO-msln, MSTO-CXCL16, MSTO-msln- CXCL16, and Suit-2 cell lines were used. After the 48 hour incubation, TFP T cells were added to the top well at 9: 1, 3 : 1, and 1 : 1 T cell Tumor cell ratios (E:T). All plates were set up in duplicate to use one plate for staining and flow cytometry, and the other for cytotoxicity. Four hours after the TFP T cells were added, the insert was removed. Staining and cytotoxicity assays were performed twenty-four hours after the insert was removed. Results are provided in FIG. 13 and FIG. 14. FIG. 13 shows that the CXCR6 expressing TFP T cells migrated in response to the presence of MSTO-CXCL16 cells. As shown in FIG. 14A-14D, CXCR6 expressing TFP T cells migrated to and killed high CXCL16 expressing tumors (MSTO-CXCL16 and MSTO-msln- CXCL16, FIG. 14C and 14D, respectively), particularly at higher E:T. Thus, the results showed that CXCR6 expressing TFP T cells migrate to and kill high CXCL16 expressing tumors faster and more effectively than they do toward CXCL16 negative tumors; and exhibit higher levels of migration and killing against high CXCL16 expressing tumors compared to TC-210.

Example 7. Ex vivo characterization

[0987] A study was conducted to assess the activity of CXCR6 expressing TFP T cells in vivo in tumor bearing mice. Mice were implanted with MSTO-CXCL16-luc tumor cells on Day -14. At Day -1, mice were randomized into the groups shown below in Table 3. On Day 0, the T cells (or vehicle) were administered. T cells were normalized to 60% TFP expression prior to injection. Tissue analysis and T cell characterization were carried out at Days 4 and 7, with n=4 at each time point. Tumor tissues, spleens, and livers were assessed.

Table 3. Groups for in vivo study.

[0988] The percent of mouse CD45+ cells (FIG. 15A) and human CD45+ cells (FIG. 15B) was increased in the TME from mice that received CXCR6 TFP T cells, compared to mice that received NT or TC-210 T cells. Fewer human CD45+ T cells were found at Day 7 in the spleen and liver of mice that received TFP T cells (FIG. 15C and 15D). As shown in FIG. 16, at Day 7, there were more CXCR6 TFP T cells found in the TME compared to TC-210, as measured by VHH+ (TFP+) cells per mg tumor; even at Day 4, more VHH+ cells were found in the TME of recipients of CXCR6-MH1 TFP T cells. When the VHH+ cell count was normalized per tumor, the data showed that there were more CXCR6 TFP T cells in the TME compared to TC-210 at both of the Day 4 and Day 7 timepoints (FIG. 17). All T cells became enriched for VHH+ cells in the TME by Day 7, with high %VHH+ at that timepoint for all TFP groups; in peripheral tissues, by Day 7 there were fewer CXCR6 TFP cells (as measured by % VHH) compared to TC-210 (FIG. 18). CXCR6 expression on CXCR6 TFPs became downregulated in the TME compared to peripheral tissues, suggesting engagement with the CXCL16 ligand in the TME (FIG. 19). In addition, CXCR6 TFP T cells had higher expression of the proliferation marker Ki67 in the TME at day 7 compared to TC-210 (FIG. 20). Analyses of the composition of TFP T cells in the TME and peripheral tissues showed that VHH+ cells became enriched for CD8+ T cells within the TME by Day 7 (FIG. 21); and that both VHH+ CD4+ (FIG. 22A) and VHH+ CD8+ (FIG. 22B) T cells became enriched within the TME for TEM cells by Day 7.

[0989] Together, the results showed that more CD45+ cells overall, and more VHH+ cells, accumulated in the TME on Day 7 in CXCR6 TFP groups, compared to the TC-210 group; the highest number of cells in the TME was observed with the CXCR6-MH1 group. The data suggest that CXCR6 TFP T cells more readily migrate to the CXCL16 expressing tumor where they interact with the ligand, proliferate, and tend to express an effector memory phenotype, all by Day 7 after administration. The results indicated that CXCR6 TFP T cells may provide a superior anti -turn or effect in cancers associated with CXCL16 expression.

Example 8. In vivo efficacy

[0990] A study is conducted to assess the in vivo antitumor activity of CXCR6 expressing, TFP expressing T cells. Mice are implanted with CXCL16 expressing tumor cells (e.g., MSTO- CXCL16-luc). At a defined timepoint following implantation (e.g., about Day 14), mice are randomized into groups including vehicle control, non-transduced T cell recipients, TC-210 recipients, and CXCR6 TFP recipients (e.g., CXCR6-MH1, pLRPO-MHl-CXCR6, and/or pLCuS-MHl-CXCR6 as shown in Example 7). T cells are administered on or after the day of randomization. Tumors are measured every 3-4 days to determine anti -turn or efficacy of the T cells. Cell persistence is also tracked via bleeds (e.g., weekly). Tumor, liver, and spleen are collected at the end of the study to assess tumor infiltration of the T cells. The results of the study will show the in vivo efficacy of CXCR6-expressing TFP T cells.

Table 4: Exemplary CXCR6 sequences

Table 5. Exemplary Antigen Binding Domain Sequences

Table 6: Exemplary TGFBrl Switch Polypeptide Sequences

Table 7: Exemplary Dominant Negative TGFBR2 receptor Sequences

Table 8: Exemplary PD-1 Sequences

Table 9. Exemplary IL-15 or IL-15R Sequences

Table 10. Exemplary Construct Sequences

OTHER EMBODIMENTS

[0991] The disclosure set forth above may encompass multiple distinct inventions with independent utility. Although each of these inventions has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the inventions includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and/or properties as described herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed in this application, in applications claiming priority from this application, or in related applications. Such claims, whether directed to a different invention or to the same invention, and whether broader, narrower, equal, or different in scope in comparison to the original claims, also are regarded as included within the subject matter of the inventions of the present disclosure.

Claims

CLAIMS WHAT IS CLAIMED IS:

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

(a) a TCR subunit comprising:

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

(ii) a TCR transmembrane domain, and

(b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked, and a second nucleic acid sequence encoding C-X-C chemokine receptor type 6 (CXCR6) or a functional fragment thereof.

2. The recombinant nucleic acid of claim 1, wherein the TCR subunit further comprises a TCR intracellular domain.

3. The recombinant nucleic acid of claim 1 or 2, wherein the first and the second nucleic acid molecules are expressed in the same operon.

4. The recombinant nucleic acid of any one of claims 1-3, wherein the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a sequence encoding a linker.

5. The recombinant nucleic acid of claim 4, wherein the linker comprises a protease cleavage site.

6. The recombinant nucleic acid of claim 5, wherein the protease cleavage site is a 2A cleavage site.

7. The recombinant nucleic acid of claim 6, wherein the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.

8. The recombinant nucleic acid of claim 1 or 2, wherein the first nucleic acid sequence and the second nucleic acid sequence are present on different nucleic acid molecules.

9. The recombinant nucleic acid of any one of claims 1-8, wherein the CXCR6 or functional fragment thereof comprises a sequence with at least 80% sequence identity to any one selected from SEQ ID NOs: 400-402.

10. The recombinant nucleic acid of any one of claims 1-8, wherein the CXCR6 or functional fragment thereof comprises a sequence of any one selected from SEQ ID NOs: 400-402.

11. The recombinant nucleic acid of any one of claims 1-10, wherein the sequence of the recombinant nucleic acid is codon optimized. The recombinant nucleic acid of claim 11, wherein the CXCR6 or functional fragment thereof is encoded by a nucleic acid with at least 60% sequence identity to SEQ ID NO: 427. The recombinant nucleic acid of claim 11, wherein the CXCR6 or functional fragment thereof is encoded by the nucleic acid of SEQ ID NO: 427. The recombinant nucleic acid of any one of claims 1-8, wherein the CXCR6 or functional fragment thereof comprises at least one, two, three, or four extracellular domains. The recombinant nucleic acid of any one of claims 1-8, wherein the CXCR6 or functional fragment thereof comprises four extracellular domains. The recombinant nucleic acid of any one of claims 1-8 and 14-15, wherein the CXCR6 or functional fragment thereof comprises an N-terminal extracellular region comprising a sequence with at least 80% sequence identity to SEQ ID NO: 403. The recombinant nucleic acid of any one of claims 1-8 and 14-15, wherein the CXCR6 or functional fragment thereof comprises an N-terminal extracellular region comprising the sequence of SEQ ID NO: 403. The recombinant nucleic acid of any one of claims 1-8 and 14-17, wherein the CXCR6 or functional fragment thereof comprises a CXCL16-binding domain. The recombinant nucleic acid of any one of claims 1-18, wherein the CXCR6 or functional fragment thereof is associated with the cell membrane when expressed in a T cell. The recombinant nucleic acid of claim 19, wherein the CXCR6 or functional fragment thereof comprises a transmembrane region comprising at least one, two, three, four, five, six, or seven transmembrane domains. The recombinant nucleic acid of claim 20, wherein the transmembrane region comprises the sequence of any one of SEQ ID NOs 409-415, or any combination thereof. The recombinant nucleic acid of claim 20, wherein the CXCR6 or functional fragment thereof comprises a transmembrane region comprising seven transmembrane domains. The recombinant nucleic acid of any one of claims 20-22, wherein the CXCR6 or functional fragment thereof further comprises the sequence of SEQ ID NO 406, the sequence of SEQ ID NO 407, the sequence of SEQ ID NO 408, the sequence of SEQ ID NO 416, the sequence of SEQ ID NO 417, the sequence of SEQ ID NO 418, or any combination thereof. The recombinant nucleic acid of claim 20 or 22, wherein the CXCR6 or functional fragment thereof further comprises the sequence of SEQ ID NO 406, the sequence of SEQ ID NO 407, the sequence of SEQ ID NO 408, or a combination thereof; and the sequence of the sequence of SEQ ID NO 416, the sequence of SEQ ID NO 417, the sequence of SEQ ID NO 418, or any combination thereof. The recombinant nucleic acid of any one of claims 1-8 and 14-24, wherein the CXCR6 or functional fragment thereof comprises a transmembrane region comprising a sequence with at least 80% sequence identity to SEQ ID NO: 428. The recombinant nucleic acid of any one of claims 1-8 and 14-24, wherein the CXCR6 or functional fragment thereof comprises a transmembrane region comprising the sequence of SEQ ID NO: 428. The recombinant nucleic acid of any one of claims 1-8 and 14-26, wherein the CXCR6 or functional fragment thereof comprises at least one, two, three, or four cytoplasmic domains. The recombinant nucleic acid of any one of claims 1-8 and 14-26, wherein the CXCR6 or functional fragment thereof comprises four cytoplasmic domains. The recombinant nucleic acid of any one of claims 1-8 and 14-28, wherein the CXCR6 or functional fragment thereof comprises a C-terminal cytoplasmic domain comprising a sequence with at least 80% sequence identity to SEQ ID NO: 419. The recombinant nucleic acid of any one of claims 1-8 and 14-28, wherein the CXCR6 or functional fragment thereof comprises a cytoplasmic domain comprising the sequence of SEQ ID NO: 419. The recombinant nucleic acid of claims 1-30, wherein migration of a cell expressing the CXCR6 or functional fragment thereof increases in response to CXCL16. The recombinant nucleic acid of claims 31, wherein (i) a migration rate of a cell expressing the CXCR6 or functional fragment thereof increases in response to CXCL16 , (ii) the number of cells expressing the CXCR6 or functional fragment thereof that migrate to a tumor site increases in response to CXCL16, or (iii) a combination thereof. The recombinant nucleic acid of any one of claims 1-8 and 14-32, comprising a sequence encoding an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 420, SEQ ID NO: 424, SEQ ID NO: 426, or SEQ ID NO: 435. The recombinant nucleic acid of any one of claims 1-8 and 14-32, comprising a sequence encoding the sequence of SEQ ID NO: 420, SEQ ID NO: 424, SEQ ID NO: 426, or SEQ ID NO: 435. The recombinant nucleic acid of any one of claims 1-34, wherein the TFP functionally interacts with an endogenous TCR complex when expressed in a T cell. The recombinant nucleic acid of any one of claims 2-35, wherein the TCR intracellular domain comprises a stimulatory domain from an intracellular signaling domain of CD3 gamma, CD3 delta, or CD3 epsilon. The recombinant nucleic acid of any one of claims 2-36, wherein the TCR intracellular domain comprises an intracellular domain from TCR alpha, TCR beta, TCR gamma, or TCR delta. The recombinant nucleic acid of any one of claims 1-37, wherein the antigen binding domain is connected to the TCR extracellular domain by a linker sequence. The recombinant nucleic acid of claim 38, wherein the linker sequence is 120 amino acids in length or less. The recombinant nucleic acid of any one of claims 38-39, wherein the linker sequence comprises (G4S)_n, wherein G is glycine, S is serine, and n is an integer from 1 to 10. The recombinant nucleic acid of claim 40, wherein n is an integer from 1 to 4. The recombinant nucleic acid of any one of claims 2-41, 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 recombinant nucleic acid of claim 42, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR alpha. The recombinant nucleic acid of claim 42, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR beta. The recombinant nucleic acid of claim 42, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma. The recombinant nucleic acid of claim 42, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta. The recombinant nucleic acid of claim 42, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon. The recombinant nucleic acid of claim 42, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta. The recombinant nucleic acid of claim 42, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma. The recombinant nucleic acid of any one of claims 42-49, wherein all three of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. The recombinant nucleic acid of claim 50, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon. The recombinant nucleic acid of claim 50, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta. The recombinant nucleic acid of claim 50, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma. The recombinant nucleic acid of claim 50, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR alpha. The recombinant nucleic acid of claim 54, wherein the constant domain of TCR alpha is murine. The recombinant nucleic acid of claim 50, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR beta. The recombinant nucleic acid of claim 56, wherein the constant domain of TCR beta is murine. The recombinant nucleic acid of claim 50, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR gamma. The recombinant nucleic acid of claim 50, wherein the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR delta. The recombinant nucleic acid of any one of claims 1-59, wherein the antigen binding domain is a camelid antibody or binding fragment thereof. The recombinant nucleic acid of any one of claims 1-59, wherein the antigen binding domain is a murine antibody or binding fragment thereof. The recombinant nucleic acid of any one of claims 1-59, wherein the antigen binding domain is a human or humanized antibody or binding fragment thereof. The recombinant nucleic acid of any one of claims 1-62, wherein the antigen binding domain is a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain. The recombinant nucleic acid of claim 63, wherein the antigen binding domain is a single domain antibody (sdAb). The recombinant nucleic acid of claim 64, wherein the sdAb is a VH or VHH. The recombinant nucleic acid of any one of claims 1-65, wherein the antigen binding domain is selected from the group consisting of an anti-CD19 binding domain, an anti-B- cell maturation antigen (BCMA) binding domain, and an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, anti-MUC16 binding domain, an anti-Nectin-4 binding domain, an anti-GPC3 binding domain, and an anti-TROP-2 binding domain. The recombinant nucleic acid of any one of claims 1-66, wherein a T cell expressing the TFP inhibits tumor growth. The recombinant nucleic acid of any one of claims 1-67, further comprising a leader sequence. The recombinant nucleic acid of any one of claims 1-68, wherein the recombinant nucleic acid comprises a sequence encoding (i) a TFP comprising a GM-CSFRa signal peptide, an anti-MSLN scFv or VHH antibody or a fragment thereof, a linker, a CD3 epsilon intracellular signaling domain, and (ii) the CXCR6 or fragment thereof. The recombinant nucleic acid of claim 69, wherein the recombinant nucleic acid comprises (i) a sequence encoding a TFP comprising, from the N-terminus to the C-terminus, the GM-CSFRa signal peptide operatively linked to the anti-MSLN scFv or VHH antibody or fragment thereof operatively linked to the linker operatively linked to the CD3 epsilon intracellular signaling domain and (ii) a sequence encoding CXCR6 or fragment thereof, or (i) the sequence encoding CXCR6 or fragment thereof and (ii) the sequence encoding the TFP comprising the GM-CSFRa signal peptide operatively linked to the anti-MSLN scFv or VHH antibody or fragment thereof operatively linked to the linker operatively linked to the CD3 epsilon intracellular signaling domain, wherein CXCR6 and the TFP are expressed in the same operon and are separated by a cleavable linker. The recombinant nucleic acid of claim 69 or 70, wherein the linker is a A3(G4S)3LE linker. The recombinant nucleic acid of claim 70, wherein, from the N-terminus to the C- terminus, the CD3 epsilon intracellular signaling domain is operatively linked to the CXCR6 or fragment thereof via a cleavable linker or the CXCR6 or fragment thereof is operatively linked to the GM-CSFRa signal peptide via the cleavable linker. The recombinant nucleic acid of claim 70, wherein the cleavable linker is a 2A cleavage site or a furin cleavage site. The recombinant nucleic acid of claim 73, wherein the 2A cleavage site is a P2A cleavage site or a T2A cleavage site. The recombinant nucleic acid of any one of claims 1-68, wherein the recombinant nucleic acid encodes a sequence comprising the sequences of SEQ ID NOs: 421, 422, 423, and 400. The recombinant nucleic acid of claim 75, wherein the recombinant nucleic acid encodes a sequence comprising, from the N-terminus to the C-terminus, the sequence of SEQ ID NO:

421 operatively linked to the sequence of SEQ ID NO: 422 operatively linked to the sequence of SEQ ID NO: 423 operatively linked to the sequence of SEQ ID NO: 400, or the sequence of SEQ ID NO: 400 operatively linked to the sequence of SEQ ID NO: 421 operatively linked to the sequence of SEQ ID NO: 422 operatively linked to the linker operatively linked to the sequence of SEQ ID NO: 423. The recombinant nucleic acid of claim 76, wherein the recombinant nucleic acid comprises a sequence encoding, from the N-terminus to the C-terminus, the sequence of SEQ ID NO:

422 is operatively linked to the sequence of SEQ ID NO: 423 via the sequence of SEQ ID NO: 387. The recombinant nucleic acid of claim 76 or 77, wherein the recombinant nucleic acid comprises a sequence encoding, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 423 is operatively linked to the sequence of SEQ ID NO: 400 via the sequence of SEQ ID NO: 23 or the sequence of SEQ ID NO 425 operatively linked to the sequence of SEQ ID NO: 23, or the sequence of SEQ ID NO: 400 is operatively linked to the sequence of SEQ ID NO: 421 via the sequence of SEQ ID NO: 425 linked to the sequence of SEQ ID NO: 23. The recombinant nucleic acid of any one of claims 1-78, further comprising a third nucleic acid sequence. The recombinant nucleic acid of claim 79, wherein the third nucleic acid sequence encodes a switch polypeptide comprising a transforming growth factor beta receptor II (TGFBr2) extracellular domain or a functional fragment thereof. The recombinant nucleic acid of claim 80, wherein the TGFBr2 extracellular domain or functional fragment thereof comprises a sequence with at least 80% sequence identity to SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437. The recombinant nucleic acid of claim 80, wherein the TGFBr2 extracellular domain or functional fragment thereof comprises the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437. The recombinant nucleic acid of any one of claims 80-82, wherein the switch polypeptide further comprises a switch intracellular domain. The recombinant nucleic acid of claim 83, wherein the TGFBr2 extracellular domain or functional fragment thereof is operably linked to the switch intracellular domain. The recombinant nucleic acid of claim 83 or 84, wherein the switch intracellular domain comprises an intracellular domain of a costimulatory polypeptide. The recombinant nucleic acid of claim 85, wherein the costimulatory polypeptide is selected from the group consisting of CD28, 4-1BB, IL-15Ra, 0X40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, and FcyRIII. The recombinant nucleic acid of claim 86, wherein the costimulatory polypeptide is CD28. The recombinant nucleic acid of claim 86, wherein the costimulatory polypeptide is 4- 1BB. The recombinant nucleic acid of claim 86, wherein the costimulatory polypeptide is IL- 15Ra. The recombinant nucleic acid of claim 86, wherein the switch intracellular domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:273 or SEQ ID NO:277. The recombinant nucleic acid of claim 86, wherein the switch intracellular domain comprises the sequence of SEQ ID NO:273 or SEQ ID NO:277. The recombinant nucleic acid of any one of claims 80-91, wherein the switch polypeptide further comprises a switch transmembrane domain. The recombinant nucleic acid of claim 92, wherein the TGFBr2 extracellular domain or functional fragment thereof is operably linked to the switch intracellular domain via the switch transmembrane domain. The recombinant nucleic acid of claim 92 or 93, wherein the switch transmembrane domain is a TGFBr2 transmembrane domain. The recombinant nucleic acid of any one of claims 92-94, wherein the switch transmembrane domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:272. The recombinant nucleic acid of any one of claims 92-94, wherein the switch transmembrane domain comprises the sequence of SEQ ID NO:272. The recombinant nucleic acid of claim 92 or 93, wherein the switch transmembrane domain is a transmembrane domain of the costimulatory polypeptide. The recombinant nucleic acid of claim 97, wherein the switch transmembrane domain is a transmembrane domain of CD28. The recombinant nucleic acid of claim 97, wherein the switch transmembrane domain is a transmembrane domain of 4- IBB. The recombinant nucleic acid of claim 97, wherein the switch transmembrane domain is a transmembrane domain of IL-15Ra. The recombinant nucleic acid of claim 97, wherein the switch transmembrane domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:275 or SEQ ID NO:279. The recombinant nucleic acid of claim 97, wherein the switch transmembrane domain comprises the sequence of SEQ ID NO:275 or SEQ ID NO:279. The recombinant nucleic acid of any one of claims 80-102, wherein the switch polypeptide further comprises an additional intracellular domain. The recombinant nucleic acid of claim 103, wherein the additional intracellular domain is operably linked to the C-terminus of the switch intracellular domain. The recombinant nucleic acid of claim 103 or 104, wherein the additional intracellular domain comprises an intracellular domain of IL-15Ra or signaling domain thereof. The recombinant nucleic acid of any one of claims 103-105, wherein the additional intracellular domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:372 or SEQ ID NO:383. The recombinant nucleic acid of any one of claims 103-105, wherein the additional intracellular domain comprises the sequence of SEQ ID NO:372 or SEQ ID NO:383. The recombinant nucleic acid of any one of claims 80-106, wherein the switch polypeptide comprises a TGFBr2 transmembrane domain and an intracellular signaling domain of 4- 1BB. The recombinant nucleic acid of any one of claims 80-106, wherein the switch polypeptide comprises a 4- IBB transmembrane domain and an intracellular signaling domain of 4- 1BB. The recombinant nucleic acid of any one of claims 80-106, wherein the switch polypeptide comprises a TGFBr2 transmembrane domain and an intracellular signaling domain of CD28. The recombinant nucleic acid of any one of claims 80-106, wherein the switch polypeptide comprises a CD28 transmembrane domain and an intracellular signaling domain of CD28. The recombinant nucleic acid of any one of claims 80-106, wherein the switch polypeptide comprises a sequence with at least 80% sequence identity to any one selected from SEQ ID NOs: 283, 284, 285, and 286. The recombinant nucleic acid of any one of claims 80-106, wherein the switch polypeptide comprises the sequence of SEQ ID NOs: 283, 284, 285, or 286. The recombinant nucleic acid of claim 79, wherein the third nucleic acid sequence encodes a dominant negative TGFBR2 receptor or a fragment thereof. The recombinant nucleic acid of claim 114, wherein the dominant negative TGFBR2 receptor or a fragment thereof comprises a sequence with at least 80% sequence identity to SEQ ID NO: 433 or SEQ ID NO: 434. The recombinant nucleic acid of claim 114, wherein the dominant negative TGFBR2 receptor or a fragment thereof comprises the sequence of SEQ ID NO: 433 or SEQ ID NO: 434. The recombinant nucleic acid of claim 79, wherein the third nucleic acid sequence encodes an interleukin- 15 (IL- 15) polypeptide or a fragment thereof. The recombinant nucleic acid of claim 117, wherein expression of the IL-15 polypeptide or fragment thereof increases persistence of a cell expressing the IL-15 polypeptide or fragment thereof. The recombinant nucleic acid of any one of claims 117-118, wherein the IL-15 polypeptide or fragment thereof is secreted when expressed in a cell. The recombinant nucleic acid of any one of claims 117-119, wherein the IL-15 polypeptide or fragment thereof comprises a sequence with at least 80% sequence identity to SEQ ID NO: 1242 or SEQ ID NO: 1245. The recombinant nucleic acid of any one of claims 117-119, wherein the IL-15 polypeptide or fragment thereof comprises the sequence of SEQ ID NO: 1242 or SEQ ID NO: 1245. The recombinant nucleic acid of any one of claims 117-121, wherein the third nucleic acid sequence further encodes an IL- 15 receptor (IL-15R) subunit or a fragment thereof. The recombinant nucleic acid of claim 122, wherein the IL-15R subunit is IL-15R alpha (IL-15Ra). 290 The recombinant nucleic acid of claim 123, wherein the IL-15 polypeptide or fragment thereof and the IL-15Ra are operatively linked by a second linker. The recombinant nucleic acid of claim 124, wherein the second linker is not a cleavable linker. The recombinant nucleic acid of claim 125, 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 recombinant nucleic acid of claim 126, wherein n is an integer from 1 to 4. The recombinant nucleic acid of claim 127, wherein n is 3. The recombinant nucleic acid of claim 128, wherein the second linker comprises the sequence of SEQ ID NO: 1243. The recombinant nucleic acid of any one of claims 122-129, wherein the third nucleic acid sequence encodes a fusion protein comprising the IL- 15 polypeptide or fragment thereof linked to the IL-15Ra subunit. The recombinant nucleic acid of claim 130, wherein the IL- 15 polypeptide or fragment thereof is linked to N-terminus of the IL-15Ra subunit. The recombinant nucleic acid of claim 130, wherein the fusion protein comprises amino acids 30 - 162 of IL-15. The recombinant nucleic acid of claim 130, wherein the fusion protein comprises amino acids 31 - 267 of IL-15Ra. The recombinant nucleic acid of claim 130, wherein the fusion protein further comprises a sushi domain. The recombinant nucleic acid of claim 130, wherein the fusion protein comprises a sequence with at least 80% sequence identity to SEQ ID NO: 1253. The recombinant nucleic acid of claim 130, wherein the fusion protein comprises the sequence of SEQ ID NO: 1253. The recombinant nucleic acid of any one of claims 130-136, wherein the fusion protein is expressed on cell surface when expressed in a cell. The recombinant nucleic acid of any one of claims 130-136, wherein the fusion protein is secreted when expressed in a cell. The recombinant nucleic acid of claim 79, wherein the third nucleic acid sequence encodes a PD-1 polypeptide or a fragment thereof. 291 The recombinant nucleic acid of any one of claims 139, wherein the PD-1 polypeptide or fragment thereof is operably linked via its C-terminus to the N-terminus of an intracellular domain of a costimulatory polypeptide. The recombinant nucleic acid of claim 140, wherein the PD-1 polypeptide or fragment thereof is linked to the intracellular domain of the costimulatory polypeptide via a transmembrane domain of PD-1. The recombinant nucleic acid of claim 140 or 141, wherein the costimulatory polypeptide is chosen from a group comprising 0X40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), 4- 1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, and FcyRIII. The recombinant nucleic acid of claim 140 or 141, wherein the intracellular domain of the costimulatory polypeptide comprises at least a portion of CD28. The recombinant nucleic acid of claim 140, wherein an extracellular domain and a transmembrane domain of PD-1 are linked to an intracellular domain of CD28. The recombinant nucleic acid of claim 144, wherein the third nucleic acid sequence encodes a fusion protein comprising the extracellular domain and the transmembrane domain of PD-1 are linked to the intracellular domain of CD28. The recombinant nucleic acid of claim 145, wherein the fusion protein comprises a sequence with at least 80% sequence identity to SEQ ID NO: 1239 or SEQ ID NO: 1244. The recombinant nucleic acid of claim 145, wherein the fusion protein comprises the sequence of SEQ ID NO: 1239 or SEQ ID NO: 1244. The recombinant nucleic acid of claim 139, wherein the third nucleic acid sequence encodes a fusion protein comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15Ra. The recombinant nucleic acid of claim 148, wherein the fusion protein comprises a sequence with at least 80% sequence identity to SEQ ID NO: 1254 or SEQ ID NO: 1262. The recombinant nucleic acid of claim 148, wherein the fusion protein comprises the sequence of SEQ ID NO: 1254 or SEQ ID NO: 1262. The recombinant nucleic acid of any one of claims 79-150, wherein the third nucleic acid sequence is operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof. The recombinant nucleic acid of claim 151, wherein the third nucleic acid sequence is operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof by a sequence encoding a third linker. 292 The recombinant nucleic acid of claim 152, wherein the third linker comprises a protease cleavage site. The recombinant nucleic acid of claim 153, wherein the protease cleavage site is a 2A cleavage site. The recombinant nucleic acid of claim 154, wherein the 2A cleavage site is a T2A cleavage site or a P2A cleavage site. The recombinant nucleic acid of any one of claims 79-150, wherein the third nucleic acid sequence and the first nucleic acid sequence, the third nucleic acid sequence and the second nucleic acid sequence, or the third nucleic acid sequence, the first nucleic acid sequence, and the second nucleic acid sequence are present on different nucleic acid molecules. The recombinant nucleic acid of any one of claims 79-156, further comprising a fourth nucleic acid sequence. The recombinant nucleic acid of claim 157, wherein the fourth nucleic acid sequence encodes a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof, a dominant negative TGFBR2 receptor or a fragment thereof, an IL- 15 polypeptide or a fragment thereof, an IL-15 polypeptide or a fragment thereof operatively linked to an IL-15R subunit or a fragment thereof, a PD-1 polypeptide or a fragment thereof, or a fusion protein comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15Ra. The recombinant nucleic acid of any one of claims 157-158, wherein the fourth nucleic acid sequence encodes a polypeptide having a sequence with at least 80% sequence identity to any one selected from SEQ ID NOs: 283, 284, 285, 286, 433, 434, 1242, 1245, 1253, 1239, 1244, 1254, and 1262. The recombinant nucleic acid of any one of claims 157-158, wherein the fourth nucleic acid sequence encodes a polypeptide having the sequence of SEQ ID NOs: 283, 284, 285, 286, 433, 434, 1242, 1245, 1253, 1239, 1244, 1254, or 1262. The recombinant nucleic acid of any one of claims 157-160, wherein the fourth nucleic acid sequence encodes a polypeptide different from a polypeptide encoded by the third nucleic acid sequence. The recombinant nucleic acid of any one of claims 157-161, wherein the fourth nucleic acid sequence is operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, or a combination thereof. 293 The recombinant nucleic acid of claim 162, wherein the third nucleic acid sequence is operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence by a sequence encoding a fourth linker. The recombinant nucleic acid of claim 163, wherein the fourth linker comprises a protease cleavage site. The recombinant nucleic acid of claim 164, wherein the protease cleavage site is a 2A cleavage site. The recombinant nucleic acid of claim 165, wherein the 2A cleavage site is a T2A cleavage site or a P2A cleavage site. The recombinant nucleic acid of any one of claims 157-161, wherein the fourth nucleic acid sequence and the first nucleic acid sequence, the fourth nucleic acid sequence and the second nucleic acid sequence, the fourth nucleic acid sequence and the third nucleic acid sequence, or the fourth nucleic acid sequence, the first nucleic acid sequence, and the second nucleic acid sequence, and the third nucleic acid sequence are present on different nucleic acid molecules. The recombinant nucleic acid of any one of claims 1-167, wherein the recombinant nucleic acid is selected from the group consisting of a DNA and an RNA. The recombinant nucleic acid of claim 168, wherein the recombinant nucleic acid is an mRNA. The recombinant nucleic acid of claim 168, wherein the recombinant nucleic acid is a circRNA. The recombinant nucleic acid of any one of claims 1-170, wherein the recombinant nucleic acid comprises a nucleotide analog. The recombinant nucleic acid of claim 171, wherein the nucleotide analog is selected from the group consisting of 2’-O-methyl, 2’-O-methoxyethyl (2’-0-M0E), 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. The recombinant nucleic acid of any one of claims 1-172, further comprising a promoter. The recombinant nucleic acid of any one of claims 1-173, wherein the recombinant nucleic 294 acid is an in vitro transcribed nucleic acid. The recombinant nucleic acid of any one of claims 1-174, further comprising a sequence encoding a poly(A) tail. The recombinant nucleic acid of any one of claims 1-175, further comprising a 3’UTR sequence. A polypeptide encoded by the recombinant nucleic acid of any one of claims 1-176. A vector comprising a recombinant nucleic acid of any one of claims 1-176. The vector of claim 178, wherein the vector is a lentiviral vector. A cell comprising the recombinant nucleic acid of any one of claims 1-176, the polypeptide of claim 177, or the vector of claim 178 or 179. The cell of claim 180, wherein the cell is a T cell. The cell of claim 181, wherein the T cell is a human T cell. The cell of claim 181 or 182, wherein the T cell is a CD8+ or CD4+ T cell. The cell of claim 181, wherein the T cell is a human aP T cell. The cell of claim 181, wherein the T cell is a human y6 T cell. The cell of claim 180, wherein the cell is a human NKT cell. The cell of any one of claims 180-186, wherein the cell is an allogeneic cell or an autologous cell. The cell of any one of claims 180-187, wherein the cell has increased anti-tumor efficacy compared to the anti-tumor efficacy of a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence. The cell of any one of claims 180-188, wherein the cell has enhanced migration compared to migration of a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence. The cell of claim 189, wherein in response to CXCL16 (i) the cell has a higher migration rate compared to a migration rate of a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence, (ii) more of the cells migrate to a tumor in response to CXCL16 compared to the number of cells comprising the first nucleic acid sequence and not comprising the second nucleic acid sequence that migrate to a tumor, or (iii) a combination thereof. The cell of any one of claims 180-190, wherein the cell has enhanced tumor lysis activity compared to a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence. 295 The cell of any one of claims 180-191, wherein the cell has increased cytokine production compared to a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence. The cell of any one of claims 180-192, wherein the cell comprises a population of cells. The cell of claim 193, wherein the population of cells comprises at least lxl0^A5 cells or at least lxlO^A6 cells. A pharmaceutical composition comprising the cell of any one of claims 180-194 and a pharmaceutically acceptable carrier. A method of increasing an activity of a cell expressing a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP), the method comprising expressing CXCR6 or a functional fragment thereof in the cell: wherein the TFP comprises:

(a) a TCR subunit comprising:

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

(ii) a TCR transmembrane domain, and

(b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked. The method of claim 196, wherein the cell is the cell of any one of claims 180-194. The method of claim 196 or 197, wherein the cell has enhanced migration compared to migration of a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. The method of claim 198, wherein (i) the migration rate of the cell in response to CXCL16 is faster compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof, (ii) more number of the cell migrates in response to CXCL16 compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof, or (iii) a combination thereof. The method of any one of claims 196-199, wherein the cell has enhanced tumor lysis activity compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. The method of any one of claims 196-200, wherein the cell has increased cytokine production compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof. A method of enhancing migration of a cell expressing a recombinant nucleic acid 296 comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP), the method comprising expressing CXCR6 or a functional fragment thereof in the cell: wherein the TFP comprises:

(a) a TCR subunit comprising:

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

(ii) a TCR transmembrane domain, and

(b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked. The method of claim 202, wherein the cell is the cell of any one of claims 180-194. The method of claim 202 or 203, wherein (i) the migration rate of the cell in response to CXCL16 is faster compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof, (ii) more number of the cell migrates in response to CXCL16 compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof, or (iii) a combination thereof. A method of enhancing tumor lysis activity of a cell expressing a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP), the method comprising expressing CXCR6 or a functional fragment thereof in the cell: wherein the TFP comprises:

(a) a TCR subunit comprising:

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

(ii) a TCR transmembrane domain, and

(b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked. The method of claim 205, wherein the cell is the cell of any one of claims 180-194. A method of increasing cytokine production by a cell expressing a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP), the method comprising expressing CXCR6 or a functional fragment thereof in the cell: wherein the TFP comprises:

(a) a TCR subunit comprising:

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

(ii) a TCR transmembrane domain, and

(b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked. 297 The method of claim 207, wherein the cell is the cell of any one of claims 180-194. 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 composition of claim 195. The method of claim 209, wherein 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. The method of claim 209 or 210, wherein the cancer is a hematologic cancer selected from the group consisting of B-cell acute lymphoid leukemia (B-ALL), T cell acute lymphoid leukemia (T-ALL), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell-follicular lymphoma, large cell- follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin’s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and preleukemia. The method of claim 209 or 210, wherein the cancer is mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, thyroid cancer, bladder cancer, ureter cancer, kidney cancer, endometrial cancer, esophageal cancer, gastric cancer, thymic carcinoma or cholangiocarcinoma. The method of any one of claims 209-212, wherein the subject is a human.