Soluble TCRS and methods of use thereof

Abstract

The present disclosure provides a therapy with soluble T cell receptors (TCRs) and cells comprising chimeric antigen receptors (CAR) including compositions, polynucleotides encoding the compositions and methods of use thereof. The methods and compositions provided herein can be used for the treatment of cancer.

Description

SOLUBLE TCRS AND METHODS OF USE THEREOFCROSS-REFERENCE

[0001] This application claims priority to U.S. Provisional Application No. 63 / 635,216, filed April 17, 2024, and U.S. Provisional Application No. 63 / 721,039, filed November 15, 2024, each of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Cancer is a leading cause of death worldwide, accounting for nearly one in six deaths globally. While cell-based immunotherapies using T cells expressing chimeric antigen receptors (CARs) have led to advances in treatment options for cancer, they may be associated with safety issues and lack of controllable activations. There is a need for methods and compositions to improve the cell-based immunotherapies using T cells expressing CARs.SUMMARY

[0003] Recognized herein is a need for an improved cell therapy with improved efficacy, specificity, and / or universal applicability.

[0004] Provided herein is a composition comprising a soluble T-cell receptor (TCR) having at least one tag (tag-sTCR), or a polynucleotide encoding the tag-sTCR, wherein the tag- sTCR binds to a peptide:MHC complex of a target cell.

[0005] In some embodiments, the composition further comprises a chimeric receptor (CAR) or polynucleotide encoding the CAR, and wherein the CAR comprises (i) an extracellular domain comprising an anti-tag binding domain that binds to the at least one tag of the tag- sTCR, (ii) a transmembrane domain, and (iii) an intracellular domain.

[0006] In some embodiments, the CAR is expressed on a surface of an immune cell.

[0007] Also provided herein is a composition comprising a soluble TCR having at least one tag (tag-sTCR) or a polynucleotide encoding the tag-sTCR; and an immune cell comprising a chimeric receptor (CAR) or polynucleotide encoding the CAR, wherein the CAR comprises (i) an extracellular domain comprising an anti-tag binding domain that binds to the at least one tag of the tag-sTCR, (ii) a transmembrane domain, and (iii) an intracellular domain.

[0008] In some embodiments, the tag-sTCR binds to a peptide:MHC complex of a target cell.

[0009] In some embodiments, the soluble TCR comprises a peptide:MHC complex engager comprising a TCR alpha variable domain and a TCR beta variable domain.

[0010] In some embodiments, the soluble TCR is not a single chain TCR.

[0011] In some embodiments, the soluble TCR does not comprise a constant region of a TCR.

[0012] In some embodiments, the soluble TCR is a single chain TCR, and wherein the TCR alpha variable domain is operably linked to the TCR beta variable domain.

[0013] In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the TCR alpha variable domain operably linked to the TCR beta variable domain.

[0014] In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the TCR beta variable domain operably linked to the TCR alpha variable domain.

[0015] In some embodiments, the tag-sTCR is a fusion protein, and wherein the at least one tag is operably linked to the soluble TCR.

[0016] In some embodiments, the at least one tag is operably linked to the N-terminus of the soluble TCR.

[0017] In some embodiments, the at least one tag is operably linked to the C-terminus of the soluble TCR.

[0018] In some embodiments, the soluble TCR is a single chain TCR, and wherein the TCR comprises, from N-terminus to C-terminus, the TCR beta variable domain, the TCR beta constant domain, the TCR alpha variable domain, and the TCR alpha constant domain.

[0019] In some embodiments, the at least one tag is operably linked to the N-terminus of the single chain TCR.

[0020] In some embodiments, the soluble TCR comprises a linker.

[0021] In some embodiments, the linker is operably linked to the TCR alpha constant domain and the TCR alpha variable domain.

[0022] In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

[0023] In some embodiments, n is 7.

[0024] In some embodiments, the soluble TCR comprises an additional tag operably linked to the C-terminus of the soluble TCR.

[0025] In some embodiments, the additional tag is a lOXHIS-sortag operably linked to the C- terminus of the soluble TCR.

[0026] In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the at least one tag, the TCR beta variable domain, the TCR beta constant domain, the linker, the TCR alpha variable domain, the TCR alpha constant domain, and the additional tag.

[0027] In some embodiments, the soluble TCR is a single chain TCR, and wherein the TCR comprises, from N-terminus to C-terminus, the TCR alpha variable domain, the TCR alpha constant domain, the TCR beta variable domain, and the TCR beta constant domain.

[0028] In some embodiments, the at least one tag is operably linked to the TCR beta variable domain.

[0029] In some embodiments, the soluble TCR comprises a linker.

[0030] In some embodiments, the linker is operably linked to the at least one tag and the TCR beta constant domain.

[0031] In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

[0032] In some embodiments, n is 4.

[0033] In some embodiments, the soluble TCR comprises an additional tag operably linked to the C-terminus of the soluble TCR.

[0034] In some embodiments, the additional tag is a lOXHIS-sortag operably linked to the C- terminus of the soluble TCR.

[0035] In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the TCR alpha variable domain, the TCR alpha constant domain, the linker, the at least one tag, the TCR beta variable region, the TCR beta constant region, and the additional tag.

[0036] In some embodiments, the soluble TCR comprises one or more disulfide bridges between the TCR alpha constant domain and the TCR beta constant domain.

[0037] In some embodiments, the soluble TCR comprises a modification comprising removal of one or more N-glycosylation sites in the TCR alpha constant domain and / or TCR beta constant domain.

[0038] In some embodiments, the TCR alpha variable domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR alpha variable domain sequences of Tables 5, 6, 8, 11, and 12.

[0039] In some embodiments, the TCR beta variable domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR beta variable domain sequences of Tables 5, 6, 8, 11, and 12.

[0040] In some embodiments, the TCR alpha constant domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR alpha constant domain sequences of Tables 5, 6, 8, 11, and 12.

[0041] In some embodiments, the TCR beta constant domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR beta constant domain sequences of Tables 5, 6, 8, 11, and 12.

[0042] In some embodiments, the additional tag comprises a sequence of SEQ ID NO: 131.

[0043] In some embodiments, the at least one tag binds to the anti-tag binding domain of the CAR.

[0044] In some embodiments, the anti-tag binding domain is an antibody or antigen binding fragment thereof.

[0045] In some embodiments, the at least one tag is not an affinity tag.

[0046] In some embodiments, the at least one tag does not comprise a fluorophore.

[0047] In some embodiments, the tag-sTCR does not comprise an antibody or any fragment thereof.

[0048] In some embodiments, the tag-sTCR does not comprise an scFv.

[0049] In some embodiments, the at least one tag is a polypeptide that is absent in eukaryotic systems.

[0050] In some embodiments, the at least one tag comprises less than 200, less than 100, less than 50, less than 30 or less than 20 amino acids.

[0051] In some embodiments, the at least one tag comprises at least 5, at least 10, at least 15, at least 20, at least 50, at least 100 or more amino acids.

[0052] In some embodiments, the at least one tag does not comprise modified amino acids or unnatural amino acids.

[0053] In some embodiments, all amino acids of the at least one tag are non-modified amino acids with natural peptide bonds.

[0054] In some embodiments, the at least one tag forms a stable alpha-helical structure.

[0055] In some embodiments, the at least one tag is hydrophilic and neutral at physiological pH.

[0056] In some embodiments, the at least one tag comprises a sequence of SEQ ID NO: 126, or a sequence that comprises less than 3 amino acids differing from the above within the sequence.

[0057] In some embodiments, the at least one tag further comprises 1, 2, 3, 4 or more amino acids on the N terminus of the sequence, or the C terminus of the sequence, or both the N terminus and the C terminus of the sequence.

[0058] In some embodiments, the at least one tag is recognized by the anti-tag binding domain of the CAR with a 'j of less than 1,000 nM, less than 500 nM, less than 250 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM or less than 10 nM.

[0059] In some embodiments, the peptide:MHC complex engager specifically binds to an endogenous disease specific antigen.

[0060] In some embodiments, the peptide:MHC complex engager specifically binds to a cancer antigen.

[0061] In some embodiments, the cancer antigen is a neoantigen, a tumor associated antigen, or a viral antigen.

[0062] In some embodiments, the peptide:MHC complex engager binds to the peptide:MHC complex comprising an epitope from the endogenous disease specific antigen that is presented on the surface of the target cell.

[0063] In some embodiments, the target cell is a cancer cell.

[0064] In some embodiments, the peptide:MHC complex engager binds to the peptide:MHC complex with a Kd of less than 1,000 nM, less than 500 nM, less than 250 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM or less than 10 nM.

[0065] In some embodiments, an MHC of the peptide:MHC complex is a class I MHC.

[0066] In some embodiments, an MHC of the peptideMHC complex is a class II MHC.

[0067] In some embodiments, the epitope is derived from a RAS polypeptide or a fragment thereof.

[0068] In some embodiments, the RAS polypeptide is a mutated RAS polypeptide.

[0069] In some embodiments, the epitope derived from the RAS polypeptide or a fragment thereof comprises a G12C, G12V, G12D, or G12R mutation.

[0070] In some embodiments, the peptide:MHC complex comprises an epitope having a sequence selected from the group consisting of SEQ ID NOs: 199-205, 359, and 432.

[0071] In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NOs: 202 or 432.

[0072] In some embodiments, an MHC of the peptideMHC complex comprising the epitope derived from the RAS polypeptide or a fragment thereof comprises an MHC encoded by an HLA-A*02:01 allele or an HLA-C*08:02 allele.

[0073] In some embodiments, the epitope is derived from a GATA3 polypeptide or a fragment thereof.

[0074] In some embodiments, the GATA3 polypeptide is a mutated GATA3 polypeptide.

[0075] In some embodiments, the GATA3 polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 485-487.

[0076] In some embodiments, the epitope derived from the GATA3 polypeptide or a fragment thereof comprises the epitope having a sequence selected from the group consisting of an epitope from SEQ ID NO: 485, an epitope from SEQ ID NO: 486, and an epitope from SEQ ID NO: 487.

[0077] In some embodiments, the epitope comprises a sequence MLTGPPARV (SEQ ID NO: 15).

[0078] In some embodiments, the epitope is derived from a Human Papillomavirus (HPV) protein.

[0079] In some embodiments, the HPV protein is a HPV16 E7 protein.

[0080] In some embodiments, the epitope comprises a sequence of SEQ ID NO: 458.

[0081] In some embodiments, the single chain TCR comprises a linker between the TCR alpha variable domain and the TCR beta variable domain.

[0082] In some embodiments, the linker is a polypeptide linker.

[0083] In some embodiments, the linker comprises the at least one tag sequence.

[0084] In some embodiments, the linker comprises a sequence selected from the group consisting of SEQ ID NOs: 125, 128, 135, and 488.

[0085] In some embodiments, the soluble TCR is a single chain TCR, and wherein the single chain TCR comprises, from N terminus to C terminus, the TCR alpha variable domain, the TCR alpha constant domain, a first linker, a first tag, a second linker, a second tag, the TCR beta variable domain, and the TCR beta constant domain.

[0086] In some embodiments, the first tag or the second tag comprises a sequence of SEQ ID NO: 126.

[0087] In some embodiments, the first tag and the second tag each comprises a sequence of SEQ ID NO: 126.

[0088] In some embodiments, the first linker or the second linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

[0089] In some embodiments, n is 2 or 4.

[0090] In some embodiments, the first linker comprises a sequence of (GGGGS)4, and the second linker comprises a sequence of GGGGS.

[0091] In some embodiments, the first linker comprises a sequence of (GGGGS)2, and the second linker comprises a sequence of GGGGS.

[0092] In some embodiments, the soluble TCR comprises a sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 552, 556, and 557.

[0093] In some embodiments, the TCR alpha variable domain and the TCR beta variable domain are on two separate polypeptide chains comprising a first polypeptide chain and a second polypeptide chain.

[0094] In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 138, 541, 543, 544, 545, 547, 548, and 550.

[0095] In some embodiments, the first polypeptide chain comprises a first dimerizing domain fused to the TCR alpha variable domain, and the second polypeptide chain comprises a second dimerizing domain fused to the TCR beta variable domain.

[0096] In some embodiments, the first dimerizing domain or the second dimerizing domain comprises at least 2, at least 5, at least 10 or more amino acids of a TCR extracellular domain adjacent to a TCR transmembrane domain.

[0097] In some embodiments, the first dimerizing domain comprises a sequence as set forth in SEQ ID NO: 425.

[0098] In some embodiments, the second dimerizing domain comprises a sequence as set forth in SEQ ID NO: 431.

[0099] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain and the first dimerizing domain; and the second polypeptide comprises, from N terminus to C terminus, the TCR beta variable domain and the second dimerizing domain.

[0100] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, a first peptide linker, and the first dimerizing domain; and the second polypeptide comprises, from N terminus to C terminus, the TCR beta variable domain, a second peptide linker, and the second dimerizing domain.

[0101] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain and the TCR alpha variable domain, and the second polypeptide comprises, from N terminus to C terminus, the second dimerizing domain and the TCR beta variable domain.

[0102] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain, a first peptide linker, and the TCR alpha variabledomain, and the second polypeptide comprises, from N terminus to C terminus, the second dimerizing domain, a second peptide linker, and the TCR beta variable domain.

[0103] In some embodiments, the first dimerizing domain comprises a TCR alpha constant (TRAC) domain or portion thereof; and the second dimerizing domain comprises a TCR beta constant (TRBC) domain or portion thereof.

[0104] In some embodiments, the first dimerizing domain and the second dimerizing domain are linked by one or more disulfide bridges.

[0105] In some embodiments, the first dimerizing domain and the second dimerizing domain are linked by a single disulfide bridge.

[0106] In some embodiments, the first polypeptide chain comprises the at least one tag.

[0107] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, and the at least one tag.

[0108] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, a linker, and the at least one tag.

[0109] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the TCR alpha variable domain, and the first dimerizing domain.

[0110] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, a linker, the TCR alpha variable domain, and the first dimerizing domain.[oni] In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

[0112] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain, the TCR alpha variable domain, and the at least one tag.

[0113] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the first dimerizing domain, and the TCR alpha variable domain.

[0114] In some embodiments, the second polypeptide chain comprises the at least one tag.

[0115] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the TCR beta variable domain, and the second dimerizing domain.

[0116] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, a linker, the TCR beta variable domain, and the second dimerizing domain.

[0117] In some embodiments, second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, and the at least one tag.

[0118] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, a linker, and the at least one tag.

[0119] In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

[0120] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the second dimerizing domain, the TCR beta variable domain, and the at least one tag.

[0121] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the second dimerizing domain, and the TCR beta variable domain.

[0122] In some embodiments, the at least one tag comprises two or more tags.

[0123] In some embodiments, the at least one tag comprises two tags.

[0124] In some embodiments, the at least one tag comprises three tags.

[0125] In some embodiments, each tag of the two or more tags comprises a sequence of SEQID NO: 126.

[0126] In some embodiments, the two or more tags are operably linked by a linker having a sequence of (GGGGS)n, where n is an integer from 1 to 10.

[0127] In some embodiments, the two or more tags are operably linked by a linker having a sequence of (GGGGS)4.

[0128] In some embodiments, the two or more tags are linked to the same first polypeptide chain or the same second polypeptide chain.

[0129] In some embodiments, the two or more tags are linked to different polypeptide chains.

[0130] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, and the two or more tags operably linked.

[0131] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, a first linker, a first tag, a second linker, and a second tag.

[0132] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the two or more tags operably linked, the TCR alpha variable domain, and the first dimerizing domain.

[0133] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, a first tag, a first linker, a second tag, a second linker, the TCR alpha variable domain, and the first dimerizing domain.

[0134] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, and the two or more tags operably linked.

[0135] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, a first linker, a first tag, a second linker, and a second tag.

[0136] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the two or more tags operably linked, the TCR beta variable domain, and the second dimerizing domain.

[0137] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, a first tag, a first linker, a second tag, a second linker, the TCR beta variable domain, and the second dimerizing domain.

[0138] In some embodiments, the first tag and the second tag comprise the same sequence.

[0139] In some embodiments, the sequence comprises a sequence of SEQ ID NO: 126.

[0140] In some embodiments, the first linker or the second linker comprises a sequence of (GGGGS)n, where n is an integer from 1 to 10.

[0141] In some embodiments, the soluble TCR comprises two or more soluble TCRs, wherein each soluble TCR of the two or more soluble TCRs recognizes a different peptide:MHC complex, wherein (i) the different peptide:MHC complexes are on the same target cell or different target cells, (ii) the different peptide:MHC complexes comprisedifferent epitope sequences, and / or (iii) the different peptide:MHC complexes comprise different MHC molecules encoded by different HLA alleles.

[0142] In some embodiments, the different peptide:MHC complexes comprise different epitope sequences presented by the same MHC molecule encoded by the same HLA allele.

[0143] In some embodiments, the different peptide:MHC complexes comprise different epitope sequences presented by different MHC molecules encoded by different HLA alleles.

[0144] In some embodiments, the different peptide:MHC complexes comprise the same epitope sequence presented by different MHC molecule encoded by different HLA alleles.

[0145] In some embodiments, the different peptide:MHC complexes are on the same target cell.

[0146] In some embodiments, the different peptide:MHC complexes are on different target cells.

[0147] In some embodiments, the soluble TCR comprises the TCR beta variable domain comprising a complementarity determining region 3 (CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 430, 435, 445, and 456.

[0148] In some embodiments, the TCR beta variable domain comprises an amino acid sequence with at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 427, 434, 442, and 453.

[0149] In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence as set forth in SEQ ID NOs: 428 or 443 and a complementarity determining region 2 (CDR2) having an amino acid sequence as set forth in SEQ ID NOs: 429 or 444.

[0150] In some embodiments, the soluble TCR comprises the TCR alpha variable domain comprising a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 422, 438, and 448, the CDR2 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 423, 239, and 449, and the CDR3 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 424, 440, and 450.

[0151] In some embodiments, the TCR alpha variable domain comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:421, 437, and 447.

[0152] In some embodiments, the soluble TCR comprises (a) the TCR beta variable domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 427, 434,442, and 453, or an amino acid sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 427, 434, 442, and 453, and (b) the TCR alpha variable domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 421, 437, and 447, or an amino acid sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 421, 437, and 447.

[0153] In some embodiments, the soluble TCR binds to the peptide:MHC complex having an epitope sequence selected from the group consisting of SEQ ID NOs: 199-205, 359, 432, and 15 in complex with an MHC molecule encoded by an HLA-A*02:01 allele or an HLA- C*08:02 allele.

[0154] In some embodiments, the tag-sTCR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 121, 127, 130, 534, and 134.

[0155] In some embodiments, the polynucleotide encoding the tag-sTCR comprises a sequence selected from the group consisting of SEQ ID NOs: 141-148 and 459-475.

[0156] Also provided herein is a cell comprising a chimeric antigen receptor (CAR) or a polynucleotide encoding the CAR, wherein the CAR comprises (i) an extracellular domain comprising a domain that binds to an ALFA tag (ii) a transmembrane domain, and (iii) an intracellular domain, and wherein the anti-tag binding domain comprises a CDR1 region comprising an amino acid sequence of SEQ ID NO: 477, a CDR2 region comprising an amino acid sequence of SEQ ID NO: 478, and a CDR3 region comprising an amino acid sequence of SEQ ID NO: 479.

[0157] In some embodiments, the transmembrane domain is derived from CD28, CD3s, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, IL2R beta, IL2R gamma, IL17Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C or a functional variant thereof.

[0158] In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain.

[0159] In some embodiments, the extracellular domain is operably linked to the transmembrane domain by a hinge domain.

[0160] In some embodiments, the hinge domain is a CD8a hinge domain.

[0161] In some embodiments, the intracellular domain comprises a primary cytoplasmic signaling sequence derived from CD3 combined with a costimulatory signaling region.

[0162] In some embodiments, the costimulatory signaling region is derived from CD28, 0X40, 4-1BB, ICOS or a functional variant thereof.

[0163] In some embodiments, the extracellular domain comprises an antibody, a functional antibody fragment, a single chain variable fragment (scFv), an Fab, a single-domain antibody (sdAb), a nanobody, a VH domain, a VL domain, a VNAR domain, a VHH domain, a bispecific antibody, a diabody, or a functional fragment or a combination thereof.

[0164] In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain and wherein the CD8a transmembrane domain has a sequence of SEQ ID NO: 480.

[0165] In some embodiments, the extracellular domain is operably linked to the transmembrane domain by a hinge domain and wherein the hinge domain is a CD8a hinge domain having a sequence of SEQ ID NO: 490.

[0166] In some embodiments, the intracellular domain comprises a primary cytoplasmic signaling sequence derived from CD3^ having a sequence of SEQ ID NO: 482 combined with a costimulatory signaling region derived from 4-1BB having a sequence of SEQ ID NO: 481.

[0167] In some embodiments, the CAR comprises a sequence with at least 85% identity to SEQ ID NO: 476.

[0168] In some embodiments, the polynucleotide encoding the CAR comprises a sequence with at least 85% identity to SEQ ID NO: 484.

[0169] In some embodiments, the cell is a T cell.

[0170] In some embodiments, the T cell is a CD4 T cell.

[0171] In some embodiments, the T cell is a CD8 T cell.

[0172] Also provided herein is a pharmaceutical composition comprising the tag-sTCR or the polynucleotide encoding the tag-sTCR of any one of the foregoing embodiments, and a pharmaceutically acceptable carrier.

[0173] Also provided herein is a pharmaceutical composition comprising the immune cell comprising the CAR or polynucleotide encoding the CAR of any one of the foregoing embodiments and a pharmaceutically acceptable carrier.

[0174] Also provided herein is use of the tag-sTCR or the polynucleotide encoding the tag- sTCR of any one of the foregoing embodiments or the immune cell comprising the CAR or polynucleotide encoding the CAR of any one of the foregoing embodiments in the manufacture of a medicament in treating a disease or a condition in a subject in need thereof.

[0175] In some embodiments, the disease or the condition is a cancer.

[0176] Also provided herein is a method of treating a disease or condition in a subject in need thereof, comprising (a) administering a composition comprising the composition of any one of the preceding embodiments; or (b) administering a therapeutically effective population of immune cells comprising the cell of any one of the foregoing embodiments; thereby treating the disease or condition in the subject.

[0177] Also provided herein is a method of preventing a disease or condition in a subject in need thereof, comprising (a) administering a composition comprising the composition of any one of the preceding embodiments; or (b) administering a therapeutically effective population of immune cells comprising the cell of any one of the preceding embodiments; thereby treating the disease or condition in the subject.

[0178] Also provided herein is a method of treating a disease or a condition in a subject in need thereof, comprising (a) administering a soluble TCR comprising a tag (tag-sTCR) or a polynucleotide encoding the tag-sTCR to a subject in need thereof, wherein the tag-sTCR binds to a target cell in the subject; and (b) administering a therapeutically-effective population of immune cells comprising a chimeric receptor (CAR) or polynucleotide encoding the CAR to the subject, wherein the CAR comprises: (i) an extracellular domain comprising an anti-tag binding domain that binds to the tag-sTCR, (ii) a transmembrane domain, and (iii) an intracellular domain comprising an intracellular signaling domain; thereby treating the disease or condition in the subject.

[0179] In some embodiments, administering in (a) is prior to, concurrently, or after administering in (b).

[0180] In some embodiments, the disease is a cancer.

[0181] In some embodiments, the cancer is a solid cancer.

[0182] In some embodiments, the cancer is a cancer expressing or diagnosed as expressing a tumor-associated antigen (TAA).

[0183] In some embodiments, the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, head and neck cancer, colorectal cancer, rectal cancer, soft-tissue sarcoma, Kaposi’s sarcoma, B- cell lymphoma (including low grade / follicular non-Hodgkin’s lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade / follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small noncleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom’s macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myeloma, Hairy cell leukemia, chronic myeloblasts leukemia, and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema, Meigs’ syndrome, and combinations thereof.

[0184] In some embodiments, the transmembrane domain is derived from CD28, CD3s, CD45, CD4, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD 137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, IL2R beta, IL2R gamma, IL17Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD 103, IT GAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or NKG2C or a functional variant thereof.

[0185] In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain.

[0186] In some embodiments, the extracellular domain is operably linked to the transmembrane domain by a hinge domain.

[0187] In some embodiments, the hinge domain is a CD8a hinge domain.

[0188] In some embodiments, the intracellular domain comprises an a primary cytoplasmic signaling sequence derived from CD3 combined with a costimulatory signaling region.

[0189] In some embodiments, the costimulatory signaling region is derived from CD28, 0X40, 4-1BB, ICOS or a functional variant thereof.

[0190] In some embodiments, the extracellular domain comprises an antibody, a functional antibody fragment, a single chain variable fragment (scFv), an Fab, a single-domain antibody (sdAb), a nanobody, a VH domain, a VL domain, a VNAR domain, a VHH domain, a bispecific antibody, a diabody, or a functional fragment or a combination thereof.

[0191] In some embodiments, the soluble TCR comprises a peptide:MHC complex engager comprising a TCR alpha variable domain and a TCR beta variable domain.

[0192] In some embodiments, the soluble TCR is not a single chain TCR.

[0193] In some embodiments, the soluble TCR does not comprise a constant region of a TCR.

[0194] In some embodiments, the at least one tag is operably linked to the soluble TCR.

[0195] In some embodiments, the soluble TCR is a single chain TCR, and wherein the TCR alpha variable domain is operably linked to the TCR beta variable domain.

[0196] In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the TCR alpha variable domain operably linked to the TCR beta variable domain.

[0197] In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the TCR beta variable domain operably linked to the TCR alpha variable domain.

[0198] In some embodiments, the at least one tag is operably linked to the N-terminus of the soluble TCR.

[0199] In some embodiments, the at least one tag is operably linked to the C-terminus of the soluble TCR.

[0200] In some embodiments, the soluble TCR is a single chain TCR, and wherein the TCR comprises, from N-terminus to C-terminus, the TCR beta variable domain, the TCR beta constant domain, a TCR alpha variable domain, and a TCR alpha constant domain.

[0201] In some embodiments, the at least one tag is operably linked to the N-terminus of the soluble TCR.

[0202] In some embodiments, the at least one tag is an ALFA-tag.

[0203] In some embodiments, the soluble TCR comprises a first linker.

[0204] In some embodiments, the at least one tag is operably linked to the TCR beta variable domain via the first linker.

[0205] In some embodiments, the first linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

[0206] In some embodiments, n is 1.

[0207] In some embodiments, the soluble TCR comprises a second linker.

[0208] In some embodiments, the second linker is operably linked to the TCR alpha constant domain and the TCR alpha variable domain.

[0209] In some embodiments, the second linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

[0210] In some embodiments, n is 7.

[0211] In some embodiments, the soluble TCR comprises an additional tag operably linked to the C-terminus of the soluble TCR.

[0212] In some embodiments, the additional tag is a lOXHIS-sortag operably linked to the C- terminus of the soluble TCR.

[0213] In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the at least one tag, the TCR beta variable domain, the TCR beta constant domain, the second linker, the TCR alpha variable domain, the TCR alpha constant domain, and the additional tag.

[0214] In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the at least one tag, the first linker, the TCR beta variable domain, the TCR beta constant domain, the second linker, the TCR alpha variable domain, the TCR alpha constant domain, and the additional tag.

[0215] In some embodiments, the soluble TCR is a single chain TCR, and wherein the TCR comprises, from N-terminus to C-terminus, the TCR alpha variable domain, a TCR alpha constant domain, the TCR beta variable domain, and a TCR beta constant domain.

[0216] In some embodiments, the at least one tag is operably linked to the TCR beta variable domain.

[0217] In some embodiments, the at least one tag is an ALFA-tag.

[0218] In some embodiments, the soluble TCR comprises a first linker.

[0219] In some embodiments, the at least one tag is operably linked to the TCR beta variable domain via the first linker.

[0220] In some embodiments, the first linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

[0221] In some embodiments, n is 1.

[0222] In some embodiments, the soluble TCR comprises a second linker.

[0223] In some embodiments, the TCR alpha constant domain is operably linked to the at least one tag via the second linker.

[0224] In some embodiments, the second linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

[0225] In some embodiments, n is 4.

[0226] In some embodiments, the soluble TCR comprises an additional tag operably linked to the C-terminus of the soluble TCR.

[0227] In some embodiments, the additional tag is a lOXHIS-sortag operably linked to the C- terminus of the soluble TCR.

[0228] In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the TCR alpha variable domain, the TCR alpha constant domain, the second linker, the at least one tag, the TCR beta variable region, the TCR beta constant region, and the additional tag.

[0229] In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the TCR alpha variable domain, the TCR alpha constant domain, the second linker, the at least one tag, the first linker, the TCR beta variable region, the TCR beta constant region, and the additional tag.

[0230] In some embodiments, the soluble TCR comprises one or more disulfide bridges between the TCR alpha constant domain and the TCR beta constant domain.

[0231] In some embodiments, the soluble TCR comprises a modification comprising removal of one or more N-glycosylation sites in the TCR alpha constant domain and / or TCR beta constant domain.

[0232] In some embodiments, removal of the one or more N-glycosylation sites comprises substituting one or more asparagine residue to one or more aspartic acid residues or one or more glutamic acid residues.

[0233] In some embodiments, the soluble TCR comprises a modification comprising removal of one or more unpaired cysteines in the TCR alpha constant domain and / or TCR beta constant domain.

[0234] In some embodiments, removal of the one or more unpaired cysteines comprises substituting one or more unpaired cysteines to one or more serine residues.

[0235] In some embodiments, the soluble TCR comprises one or more disulfide bonds in the TCR alpha constant domain and / or the TCR beta constant domain.

[0236] In some embodiments, the soluble TCR comprises one or more mutations in the TCR alpha constant domain and / or the TCR beta constant domain.

[0237] In some embodiments, the one or more mutations are one or more stability enhancing mutations.

[0238] In some embodiments, the one or more mutations in the TCR alpha constant domain comprises S139F, T150I, or A190T.

[0239] In some embodiments, the one or more mutations in the TCR beta constant domain comprises C191A, N205D, E134K, H139R, D155P, or S170D.

[0240] In some embodiments, the TCR alpha constant domain and the TCR beta constant domain comprises one or more N>Q substitutions.

[0241] In some embodiments, the soluble TCR comprises no mutations in the TCR alpha constant domain and / or the TCR beta constant domain.

[0242] In some embodiments, the soluble TCR comprises a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 489.

[0243] In some embodiments, the soluble TCR comprises a TCR alpha chain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 491.

[0244] In some embodiments, the soluble TCR comprises a TCR alpha variable domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 421.

[0245] In some embodiments, the soluble TCR comprises a TCR alpha constant domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 492.

[0246] In some embodiments, the soluble TCR comprises a TCR beta chain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 132.

[0247] In some embodiments, the soluble TCR comprises a TCR beta variable domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 427.

[0248] In some embodiments, the soluble TCR comprises a TCR beta constant domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 490.

[0249] In some embodiments, the soluble TCR comprises a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 494.

[0250] In some embodiments, the soluble TCR comprises a sequence having a sequence set forth in SEQ ID NO: 494.

[0251] In some embodiments, the soluble TCR comprises a TCR alpha chain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 496.

[0252] In some embodiments, the soluble TCR comprises a TCR alpha variable domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 421.

[0253] In some embodiments, the soluble TCR comprises a TCR alpha constant domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 492.

[0254] In some embodiments, the soluble TCR comprises a TCR beta chain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 495.

[0255] In some embodiments, the soluble TCR comprises a TCR beta variable domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 427.

[0256] In some embodiments, the soluble TCR comprises a TCR beta constant domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 490.

[0257] In some embodiments, the soluble TCR comprises a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 497.

[0258] In some embodiments, the soluble TCR comprises a sequence having a sequence set forth in SEQ ID NO: 497.

[0259] In some embodiments, the soluble TCR comprises a TCR alpha chain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 500.

[0260] In some embodiments, the soluble TCR comprises a TCR alpha variable domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 421.

[0261] In some embodiments, the soluble TCR comprises a TCR alpha constant domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 501.

[0262] In some embodiments, the soluble TCR comprises a TCR beta chain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 498.

[0263] In some embodiments, the soluble TCR comprises a TCR beta variable domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 427.

[0264] In some embodiments, the soluble TCR comprises a TCR beta constant domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 499.

[0265] In some embodiments, the TCR alpha variable domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR alpha variable domain sequences of Tables 5, 6, 8, 11, and 12.

[0266] In some embodiments, the TCR beta variable domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR beta variable domain sequences of Tables 5, 6, 8, 11, and 12.

[0267] In some embodiments, the TCR alpha constant domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR alpha constant domain sequences of Tables 5, 6, 8, 11, and 12.

[0268] In some embodiments, the TCR beta constant domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR beta constant domain sequences of Tables 5, 6, 8, 11, and 12.

[0269] In some embodiments, the additional tag comprises a sequence of SEQ ID NO: 131.

[0270] In some embodiments, the at least one tag comprises a sequence of SEQ ID NO: 126, or a sequence that comprises less than 3 amino acids differing from the above within the sequence.

[0271] In some embodiments, the soluble TCR exhibits anti-tumor cytotoxicity at a level comparable to a corresponding heterodimeric soluble TCR.

[0272] In some embodiments, the soluble TCR induces T cell proliferation at a level comparable to a corresponding heterodimeric soluble TCR.

[0273] In some embodiments, the soluble TCR upregulates expression of one or more cytotoxicity markers at a level comparable to a corresponding heterodimeric soluble TCR.

[0274] In some embodiments, the soluble TCR binds to an epitope on the surface of the target cell.

[0275] In some embodiments, the epitope is derived from a RAS polypeptide or a fragment thereof.

[0276] In some embodiments, the RAS polypeptide is a mutated RAS polypeptide.

[0277] In some embodiments, the epitope is derived from a GATA3 polypeptide or a fragment thereof.

[0278] In some embodiments, the single chain TCR comprises a linker between the TCR alpha variable domain and the TCR beta variable domain.

[0279] In some embodiments, the linker is a polypeptide linker.

[0280] In some embodiments, the linker comprises the at least one tag sequence.

[0281] In some embodiments, the linker comprises a sequence selected from the group consisting of SEQ ID NOs: 125, 128, 135, and 488.

[0282] In some embodiments, the soluble TCR is a single chain TCR, and wherein the single chain TCR comprises, from N terminus to C terminus, the TCR alpha variable domain, the TCR alpha constant domain, a first linker, a first tag, a second linker, a second tag, the TCR beta variable domain, and the TCR beta constant domain.

[0283] In some embodiments, the first tag or the second tag comprises a sequence of SEQ ID NO: 126.

[0284] In some embodiments, the first tag and the second tag each comprises a sequence of SEQ ID NO: 126.

[0285] In some embodiments, the first linker or the second linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

[0286] In some embodiments, n is 2 or 4.

[0287] In some embodiments, the first linker comprises a sequence of (GGGGS)4, and the second linker comprises a sequence of GGGGS.

[0288] In some embodiments, the first linker comprises a sequence of (GGGGS)2, and the second linker comprises a sequence of GGGGS.

[0289] In some embodiments, the soluble TCR comprises a sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 552, 556, and 557.

[0290] In some embodiments, the TCR alpha variable domain and the TCR beta variable domain are on two separate polypeptide chains comprising a first polypeptide chain and a second polypeptide chain.

[0291] In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 138, 541, 543, 544, 545, 547, 548, and 550.

[0292] In some embodiments, the first polypeptide chain comprises a first dimerizing domain fused to the TCR alpha variable domain, and the second polypeptide chain comprises a second dimerizing domain fused to the TCR beta variable domain.

[0293] In some embodiments, the first dimerizing domain or the second dimerizing domain comprises at least 2, at least 5, at least 10 or more amino acids of a TCR extracellular domain adjacent to a TCR transmembrane domain.

[0294] In some embodiments, the first dimerizing domain comprises a sequence as set forth in SEQ ID NO: 425.

[0295] In some embodiments, the second dimerizing domain comprises a sequence as set forth in SEQ ID NO: 431.

[0296] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain and the first dimerizing domain; and the second polypeptide comprises, from N terminus to C terminus, the TCR beta variable domain and the second dimerizing domain.

[0297] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, a first peptide linker, and the first dimerizing domain; and the second polypeptide comprises, from N terminus to C terminus, the TCR beta variable domain, a second peptide linker, and the second dimerizing domain.

[0298] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain and the TCR alpha variable domain, and the second polypeptide comprises, from N terminus to C terminus, the second dimerizing domain and the TCR beta variable domain.

[0299] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain, a first peptide linker, and the TCR alpha variable domain, and the second polypeptide comprises, from N terminus to C terminus, the second dimerizing domain, a second peptide linker, and the TCR beta variable domain.

[0300] In some embodiments, the first dimerizing domain comprises a TCR alpha constant (TRAC) domain or portion thereof; and the second dimerizing domain comprises a TCR beta constant (TRBC) domain or portion thereof.

[0301] In some embodiments, the first dimerizing domain and the second dimerizing domain are linked by one or more disulfide bridges.

[0302] In some embodiments, the first dimerizing domain and the second dimerizing domain are linked by a single disulfide bridge.

[0303] In some embodiments, the first polypeptide chain comprises the at least one tag.

[0304] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, and the at least one tag.

[0305] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, a linker, and the at least one tag.

[0306] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the TCR alpha variable domain, and the first dimerizing domain.

[0307] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, a linker, the TCR alpha variable domain, and the first dimerizing domain.

[0308] In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

[0309] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain, the TCR alpha variable domain, and the at least one tag.

[0310] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the tag, the first dimerizing domain, and the TCR alpha variable domain.

[0311] In some embodiments, the second polypeptide chain comprises the at least one tag.

[0312] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the TCR beta variable domain, and the second dimerizing domain.

[0313] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, a linker, the TCR beta variable domain, and the second dimerizing domain.

[0314] In some embodiments, the second polypeptide comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, and the at least one tag.

[0315] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, a linker, and the at least one tag.

[0316] In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

[0317] In some embodiments, the second polypeptide comprises, from N terminus to C terminus, the second dimerizing domain, the TCR alpha variable domain, and the at least one tag.

[0318] In some embodiments, the second polypeptide comprises, from N terminus to C terminus, the at least one tag, the second dimerizing domain, and the TCR beta variable domain.

[0319] In some embodiments, the at least one tag comprises two or more tags.

[0320] In some embodiments, the at least one tag comprises three tags.

[0321] In some embodiments, each tag of the two or more tags comprises a sequence of SEQ ID NO: 126.

[0322] In some embodiments, the two or more tags are operably linked by a linker having a sequence of (GGGGS)n, where n is an integer from 1 to 10.

[0323] In some embodiments, the two or more tags are linked to the same first polypeptide chain or the same second polypeptide chain.

[0324] In some embodiments, the two or more tags are linked to different polypeptide chains.

[0325] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, and the two or more tags operably linked.

[0326] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, a first linker, a first tag, a second linker, and a second tag.

[0327] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the two or more tags operably linked, the TCR alpha variable domain, and the first dimerizing domain.

[0328] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, a first tag, a first linker, a second tag, a second linker, the TCR alpha variable domain, and the first dimerizing domain.

[0329] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, and the two or more tags operably linked.

[0330] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, a first linker, a first tag, a second linker, and a second tag.

[0331] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the two or more tags operably linked, the TCR beta variable domain, and the second dimerizing domain.

[0332] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, a first tag, a first linker, a second tag, a second linker, the TCR beta variable domain, and the second dimerizing domain.

[0333] In some embodiments, the first tag and the second tag comprise the same sequence.

[0334] In some embodiments, the sequence comprises a sequence of SEQ ID NO: 126.

[0335] In some embodiments, the first linker or the second linker comprises a sequence of (GGGGS)n, where n is an integer from 1 to 10.

[0336] In some embodiments, the soluble TCR comprises two or more soluble TCRs, wherein each soluble TCR of the two or more soluble TCRs recognizes a different peptide:MHC complex, wherein (i) different peptide:MHC complexes are on the same target cell or different target cells, (ii) different peptide:MHC complexes comprise different epitope sequences, and / or (iii) different peptide:MHC complexes comprise different MHC molecules encoded by different HLA alleles.

[0337] In some embodiments, the different peptide:MHC complexes comprise different epitope sequences presented by the same MHC molecule encoded by the same HLA allele.

[0338] In some embodiments, the different peptide:MHC complexes comprise different epitope sequences presented by different MHC molecules encoded by different HLA alleles.

[0339] In some embodiments, the different peptide:MHC complexes comprise the same epitope sequence presented by different MHC molecule encoded by different HLA alleles.

[0340] In some embodiments, the different peptide:MHC complexes are on the same target cell.

[0341] In some embodiments, the different peptide:MHC complexes are on different target cells.INCORPORATION BY REFERENCE

[0342] 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

[0343] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0344] FIG. 1 is a cartoon depicting the structure of ALFA-tagged adaptors and interactions with T cells bearing ALFA-specific chimeric antigen receptors (CARs).

[0345] FIG. 2 is an exemplary structure of a CAR T cell binding to an ALFA-tagged soluble TCR (sTCR) that recognizes a tumor antigen presented on a tumor cell.

[0346] FIGs. 3A-3D depict exemplary soluble TCR structures. In the structures provided herein, the tail at the bottom left is the C-terminal tail of the TCR beta chain and the tail at the bottom right is the C-terminal tail of the TCR alpha chain. FIG. 3A depicts a two chain construct bearing a variable heavy and variable light chain of a ScFv specific for CD3. FIG. 3B depicts a two chain construct bearing a C-terminal ALFA tag. FIG. 3C depicts a two chain construct bearing tandem N-terminal ALFA tags. FIG. 3D depicts a two chain construct bearing an N-terminal ALFA tag.

[0347] FIG. 4 is an experimental outline to test T cell activation using the constructs depicted in FIGs. 3A-3D.

[0348] FIGs. 5A-5C depict results measuring CD69 expression according to the experiment shown in FIG. 4. FIG. 5A depicts results using an sTCR concentration of InM. FIG. 5B depicts results using an sTCR concentration of lOnM. FIG. 5C depicts results using an sTCR concentration of lOOnM.

[0349] FIG. 6 is an experimental outline to test tumor cell killing mediated by the constructs depicted in FIGs. 3A-3D.

[0350] FIG. 7 depicts tumor cell killing results from the experiment shown in FIG. 6.

[0351] FIG. 8A depicts an experiment to test the efficacy of different clones of sTCRs to induce CD69 expression.

[0352] FIG. 8B depicts the results measuring CD69 expression according to the experiment shown in FIG. 8A.

[0353] FIGs. 9A-9B depicts the results of a tumor killing assays. FIG. 9A depicts tumor killing when varying sTCR concentration. FIG. 9B depicts tumor killing when using different TCR clones.

[0354] FIG. 10 depicts quantification of killing efficacy over time of Ros9a clone sTCRs at Inm (middle) or at lOnm (right) concentrations compared to no sTCR control (left).

[0355] FIGs. 11A-11B depicts results correlating phenotypic parameters (indicated by y- axes) with efficacy of killing observed in FIG. 10A. FIG. 11A depicts correlation based on %CD4+ (upper panels), % Granzyme B+ CD4 T cells (middle panels), or %VHH+ CD4 T cells (lower panels. FIG. 11B depicts correlation based on %CD8+ (upper panels), % Granzyme B+ CD8 T cells (middle panels), or %VHH+ CD8 T cells (lower panels).

[0356] FIG. 12 is an outline of an experiment measuring the effect of peptide concentration on activation of CAR T cells.

[0357] FIGs. 13A-13B depict flow cytometry results. FIG. 13A depicts CD69 expression by CAR T cells activated according to the experiment shown in FIG. 12. FIG. 13B depicts exemplary flow cytometry plots measuring CD69 expression by CAR T cells in an experiment using A375 target cells.

[0358] FIG. 14 is an outline of an experiment measuring the effect of peptide concentration of CAR T cells at a 5: 1 effector to target ratio.

[0359] FIG. 15 depicts the killing efficacy of CAR T cells from the experiment outlined in FIG. 14.

[0360] FIG. 16 is a cartoon illustrating two different scenarios of interactions between CAR constructs and sTCRs.

[0361] FIG. 17 is an outline of an experiment testing the killing efficacy of CAR T cells when three different sTCRs at different concentrations are used.

[0362] FIGs. 18A-18B depicts the raw data of killing efficacy (target cell count) of the CAR T cells tested according to FIG. 17. FIG. 18A depicts killing efficacy for no peptide no sTCR controls (left) or with sTCR sc74 (right) at varying concentrations. FIG. 18B depicts sTCR sc32 (left) at varying concentrations or sc69 (right) at varying concentrations.

[0363] FIG. 19 is a summary of the data presented in FIGs. 18A-18B, presented as a percentage of killed cells across different peptide concentrations categorized by sTCR construct then by different sTCR concentrations.

[0364] FIG. 20 is a summary of the data presented in FIGs. 18A-18B, presented as a percentage of killed cells across different peptide concentrations categorized by sTCR concentration then by each sTCR construct.

[0365] FIGs. 21A-21B depicts flow cytometry quantifications among CD8 T cells across peptide concentrations following the experiment shown in FIG. 17. FIG. 21A depicts expression of CD25 (upper panels) or Ki67 (lower panels). FIG. 21B depicts expression of Granzyme B (upper panels) or PD-1 (lower panels).

[0366] FIGs. 22A-22B depicts flow cytometry quantifications among CD4 T cells across peptide concentrations following the experiment shown in FIG. 17. FIG. 22A depicts expression of CD25 (upper panels) or Ki67 (lower panels). FIG. 22B depicts expression of Granzyme B (upper panels) or PD-1 (lower panels).

[0367] FIG. 23 is an outline of an experiment testing the killing efficacy using target cells expressing Human Papillomavirus (HPV) as a model of cervical cancer.

[0368] FIGs. 24A-24B depicts the killing efficacy of the experiment described in FIG. 23. FIG. 24A depicts number of GFP+ cells (target cells) over time. FIG. 24B depicts percent killing efficacy within the conditions shown below the X axis.

[0369] FIG. 25 is a cartoon illustrating a “decorated” CAR T cell that expresses multiple TCRs with different specificities.

[0370] FIG. 26A depicts exemplary data from sc32 sTCR construct from the experiments shown in FIG. 19.

[0371] FIG. 26B is a quantification of tetramer staining of decorated CAR T cells at varying sTCR concentrations among both CD4 and CD8 T cells and CD3- and ALFA-specific T cells.

[0372] FIG. 27 is a cartoon illustrating a possible scenario in which multiple target / effector interactions enhance avidity of the interaction.

[0373] FIG. 28 is an experimental outline to test the efficacy of decorated multitargeted CAR T cells as measured by killing and phenotypic analysis.

[0374] FIG. 29A depicts target cell counts over time according to the experiment outlined in FIG. 28 with either KRAS or HPV target peptides at varying concentrations and using mixed or single sTCR constructs as indicated.

[0375] FIGs. 29B and 30 depicts target cell counts over time according to the experiment outlined in FIG. 28. FIG. 29B depicts cell counts when treated with either KRAS, HPV, ormixed target peptides at varying concentrations and using mixed sTCR constructs as indicated . FIG. 30 is a quantification of calculated % killing organized by sTCR condition.

[0376] FIG. 31 is an experimental outline to test the efficacy of decorated multitargeted CAR T cells as measured by killing and phenotypic analysis.

[0377] FIGs. 32A-32B are quantifications percent killing separated by CAR T cell donors across peptide concentrations. FIG. 32A depicts the results of each target peptide as a dosedependent curve. FIG. 32B depicts the results as bar graphs for each peptide concentration.

[0378] FIGs. 33A-33F depict flow cytometry data measuring expression of activation markers. FIGs. 33A and 33D depict expression of CD25. FIGs. 33B and 33E depict expression of Ki67. FIGs. 33C and 33F depict expression of Granzyme B. All data are initially gated within CD4 CAR T cells from a human donor.

[0379] FIGs. 34A-34F depict flow cytometry data measuring expression of activation markers. FIGs. 34A and 34D depict expression of CD25. FIGs. 34B and 34E depict expression of Ki67. FIGs. 34C and 34F depict expression of Granzyme B. All data are initially gated within CD4 CAR T cells from the same donor as FIGs. 33A-33F.

[0380] FIGs. 35A-35F depict flow cytometry data measuring expression of activation markers. FIGs. 35A and 35D depict expression of CD25. FIGs. 35B and 35E depict expression of Ki67. FIGs. 35C and 35F depict expression of Granzyme B. All data are initially gated within CD4 CAR T cells from a human donor.

[0381] FIGs. 36A-36F depict flow cytometry data measuring expression of activation markers. FIGs. 36A and 36D depict expression of CD25. FIGs. 36B and 36E depict expression of Ki67. FIGs. 36C and 36F depict expression of Granzyme B. All data are initially gated within CD4 CAR T cells from the same donor as FIGs. 35A-35F.

[0382] FIGs. 37A-37B illustrate a possible scenario of effector / target cell interactions. FIG. 37A illustrates multiple different TCR specificities binding to a target cells. FIG. 37B shows an exemplary measurement of the avidity of the interaction in a possibly additive manner.

[0383] FIG. 38 depicts a plasmid construct encoding an NbALFA CAR receptor.

[0384] FIGs. 39A-39D depict exemplary single chain sTCR constructs. FIG. 39A depicts a construct comprising, from N-terminus to C-terminus an ALFA tag, a TCR beta variable region, a TCR beta constant region, a linker, a TCR alpha variable region, a TCR alpha constant region and a 10X His-Sortag. FIG. 39B depicts a construct comprising, from N- terminus to C-terminus a variable light chain, a variable heavy chain, a TCR beta variable region, a TCR beta constant region, a linker, a TCR alpha variable region, a TCR alphaconstant region and a 10X His-Sortag. FIG. 39C depicts a construct comprising, from N- terminus to C-terminus an ALFA tag, a TCR beta variable region, a TCR beta constant region, a linker, a TCR alpha variable region, a TCR alpha constant region and a 10X His- Sortag. FIG. 39D depicts a construct comprising, from N-terminus to C-terminus a variable light chain, a variable heavy chain, a TCR beta variable region, a TCR beta constant region, a linker, a TCR alpha variable region, a TCR alpha constant region and a 10X His-Sortag.

[0385] FIGs. 40A-40D depict exemplary single chain sTCR constructs. FIG. 40A depicts a construct comprising, from N-terminus to C-terminus a TCR alpha variable region, a TCR alpha constant region, a linker, an ALFA tag, a TCR beta variable region, a TCR beta constant region, and a 10X His-Sortag. FIG. 40B depicts a construct comprising, from N- terminus to C-terminus a TCR alpha variable region, a TCR alpha constant region, a linker, a variable light chain, a variable heavy chain, a TCR beta variable region, a TCR beta constant region, and a 10X His-Sortag. FIG. 40C depicts a construct comprising, from N-terminus to C-terminus a TCR alpha variable region, a TCR alpha constant region, a linker, an ALFA tag, a TCR beta variable region, a TCR beta constant region, and a 10X His-Sortag. FIG. 40D depicts a construct comprising, from N-terminus to C-terminus a TCR alpha variable region, a TCR alpha constant region, a linker, a variable light chain, a variable heavy chain, a TCR beta variable region, a TCR beta constant region, and a 10X His-Sortag.

[0386] FIG. 41 depicts exemplary ALFA tagged sTCR construct.

[0387] FIG. 42 illustrates schematic of single-chain ALFA sTCR designs.

[0388] FIG. 43 is an outline of an experiment testing the killing efficacy, T cell activation, proliferation, and cytotoxicity using target cells expressing RAS G12D, single-chain ALFA sTCRs, and CAR+ T cells.

[0389] FIG. 44 depicts the result of antigen-specific tumor cell killing mediated by singlechain ALFA-tag sTCR T cells.

[0390] FIGs. 45A-45B depict the proliferation of single-chain ALFA-tag sTCR T cells. FIG. 45A shows the percentage of Ki-67+ CD4 T cells upon transduction with single-chain ALFA-tag sTCRs. FIG. 45B shows the percentage of Ki-67+ CD8 T cells upon transduction with single-chain ALFA-tag sTCRs.

[0391] FIGs. 46A-46B depict the expression of a cytotoxicity marker of single-chain ALFA- tag sTCR T cells. FIG. 46A shows the percentage of granzyme B+ CD4 T cells upon transduction with single-chain ALFA-tag sTCRs. FIG. 46B shows the percentage of granzyme B+ CD8 T cells upon transduction with single-chain ALFA-tag sTCRs.

[0392] FIGs. 47A-47B depict results measuring killing efficiency of sc82 sTCRs including the indicated TCR clones. FIG. 47A depicts results using the KRAS G12D 9-mer peptide. FIG. 47B depicts results using the KRAS G12D 10-mer peptide.

[0393] FIGs. 48A-48C depict results measuring Ki-67 and granzyme B expression of T cells cocultured with sc82 TCRs including the indicated TCR clones. FIG. 48A depict representative flow cytometry plots illustrating Ki -67 expression. FIG. 48B summarizes the expression of Ki-67 across peptide concentrations. FIG. 48C summarizes the expression of Granzyme B across peptide concentrations.

[0394] FIG. 49 is a cartoon depicting the use of a dual-ALFA tag construct to engage two CAR receptors.

[0395] FIGs. 50A-50D depict sTCR constructs. FIG. 50A depicts sc82, a single ALFA tag construct. FIG. 50B depicts sc92, a dual-ALFA tag construct, wherein the two ALFA tags are linked via a short G4S linker. FIG. 50C depicts sc93, a dual ALFA tag construct, wherein the two ALFA tags are linked via a long (G4S)4 linker. FIG. 50D depicts sc94, a triple ALFA tag construct, wherein the three ALFA tags are linked via short G4S linkers.

[0396] FIGs. 51A-51B depict results measuring killing, Ki-67 expression, and Granzyme B expression of T cells cultured with the sTCR constructs shown in FIGs. 50A-50D. FIG. 51A depicts results measuring killing efficacy. For the vehicle and 3.00E-06 groups, the bars correspond to, from left to right, no TCR, sc82, sc92, sc93, and sc94. For all other groups, the bars correspond to, from left to right, sc82, sc92, sc93, and sc94. FIG. 51B depicts results measuring Ki-67 expression (top panel) and Granzyme B expression (bottom panel).

[0397] FIGs. 52A-52B depict results measuring the killing of T cells cultured with the indicated sTCR designs and the TCR clones used. FIG. 52A depicts results using a titration of KRAS 9-mer peptide. FIG. 52B depicts results using a titration of KRAS 10-mer peptide.

[0398] FIG. 53 depicts results measuring the sTCR / CAR interaction using either singleALFA constructs or dual-ALFA constructs of the indicated TCR clones.

[0399] FIG. 54 illustrates a possible mechanism by which single- ALFA tag constructs or the dual-ALFA tag constructs interact with the CAR.

[0400] FIGs. 55A-55E depict sTCR constructs. FIG. 55A depicts sc82, a two chain single ALFA tag construct. FIG. 55B depicts scl37, a single chain single ALFA tag construct. FIG. 55C depicts sc92, a two-chain dual ALFA tag construct. FIG. 55D depicts sc 138, a single chain dual ALFA tag construct. FIG. 55E depicts sc 139, a single chain dual ALFA tag construct.

[0401] FIG. 56 depicts results measuring killing of T cells cultured with the constructs shown in FIGs. 55A-55E.

[0402] FIGs. 57A-57B depict an experiment testing the killing efficiency of a C-term ALFA construct (sc23) and a N-term ALFA construct (sc32). FIG. 57A is an experimental overview. FIG. 57B depicts the results of the killing assay.

[0403] FIG. 58 depicts results of a killing assay measuring the killing efficiency of sTCRs, using the sc82 design, including the indicated TCR clones.DETAILED DESCRIPTIONDefinitions

[0404] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0405] An antigen is a foreign substance to the body that induces an immune response. A “neoantigen” refers to a class of tumor antigens which arise from tumor-specific changes in proteins. Neoantigens encompass, but are not limited to, tumor antigens which arise from, for example, a substitution in a protein sequence, a frame shift mutation, a fusion polypeptide, an in-frame deletion, an insertion, and expression of an endogenous retroviral polypeptide.

[0406] A “neoepitope” refers to an epitope that is not present in a reference, such as a nondiseased cell, e.g., a non-cancerous cell or a germline cell, but is found in a diseased cell, e.g., a cancer cell. This includes situations where a corresponding epitope is found in a normal nondiseased cell or a germline cell but, due to one or more mutations in a diseased cell, e.g., a cancer cell, the sequence of the epitope is changed so as to result in the neoepitope.

[0407] A “mutation” refers to a change of or a difference in a nucleic acid sequence (e.g., a nucleotide substitution, addition or deletion) compared to a reference nucleic acid. A “somatic mutation” can occur in any of the cells of the body except the germ cells (sperm and egg) and are not passed on to children. These alterations can (but do not always) cause cancer or other diseases. In some embodiments, a mutation is a non-synonymous mutation. A “non- synonymous mutation” refers to a mutation, for (e.g., a nucleotide substitution), which does result in an amino acid change such as an amino acid substitution in the translation product. A “frameshift” occurs when a mutation disrupts the normal phase of a gene’s codon periodicity (also known as “reading frame”), resulting in translation of a non-native protein sequence. It is possible for different mutations in a gene to achieve the same altered reading frame.

[0408] “Antigen processing” or “processing” refers to the degradation of a polypeptide or antigen into procession products, which are fragments of said polypeptide or antigen (e.g., the degradation of a polypeptide into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by cells, for example, antigen presenting cells, to specific T cells.

[0409] An “antigen presenting cell” (APC) refers to a cell which presents peptide fragments of protein antigens in association with MHC molecules on its cell surface. The term includes professional antigen presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells) as well as other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes). In some cases, the APC can be a cancer cell.

[0410] The term “affinity” refers to a measure of the strength of binding between two members of a binding pair (e.g., a human leukocyte antigen (HLA)-binding peptide and a class I or II HLA, or a peptide-HLA complex and a T cell receptor (TCR)). KD refers to the dissociation constant between two members of a binding pair and has units of molarity. KA refers to the affinity constant between two members of a binding pair is the inverse of the dissociation constant. Affinity may be determined experimentally, for example by surface plasmon resonance (SPR) using commercially available Biacore SPR units. KOff refers to the off-rate constant of two members of a binding pair, (e.g., the off-rate constant of an HLA- binding peptide and a class I or II HLA, or a peptide-HLA complex and a TCR). Konrefers to the on-rate constant of two members of a binding pair, (e.g., the on-rate constant of an HLA- binding peptide and a class I or II HLA, or a peptide-HLA complex and a TCR).

[0411] Throughout this disclosure, “binding data” results may be expressed in terms of an “IC50.” Affinity may also be expressed as the inhibitory concentration 50 (IC50), or the concentration at which 50% of a first member of a binding pair (e.g., a peptide) is displaced. Likewise, In(ICso) refers to the natural log of the IC50. For example, an IC50 may be the concentration of a tested peptide in a binding assay at which 50% inhibition of binding of a labeled reference peptide is observed. Given the conditions in which the assays are run (e.g., limiting HLA protein concentrations and / or labeled reference peptide concentrations), these values can approximate KD values. Assays for determining binding are well known in the art and are described in detail, for example, in PCT publications WO 94 / 20127 and WO 94 / 03205, and other publications such Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); and Sette, et al., Mol. Immunol. 31 :813 (1994). Alternatively, binding can be expressed relative to binding by a reference standard peptide.Binding can also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147: 189 (1991); del Guercio et al., J. Immunol. 154:685 (1995)), cell free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21 :2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11 :2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268: 15425 (1993)); high flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and measurement of class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol. 149: 1896 (1992)).

[0412] The term “derived” when used to discuss an epitope is a synonym for “prepared.” A derived epitope can be isolated from a natural source, or it can be synthesized according to standard protocols in the art. Synthetic epitopes can comprise artificial amino acid residues “amino acid mimetics,” such as D isomers of natural occurring L amino acid residues or nonnatural amino acid residues such as cyclohexylalanine. A derived or prepared epitope can be an analog of a native epitope. The term “derived from” refers to the origin or source, and may include naturally occurring, recombinant, unpurified, purified or differentiated molecules or cells. For example, an expanded or induced antigen specific T cell may be derived from a T cell. For example, an expanded or induced antigen specific T cell may be derived from an antigen specific T cell in a biological sample. For example, a matured APC (e.g., a professional APC) may be derived from a non-matured APC (e.g., an immature APC). For example, an APC may be derived from a monocyte (e.g., a CD14+monocyte). For example, a dendritic cell may be derived from a monocyte (e.g., a CD14+monocyte). For example, an APC may be derived from a bone marrow cell.

[0413] An “epitope” is the collective features of a molecule (e.g., a peptide’s charge and primary, secondary and tertiary structure) that together form a site recognized by another molecule (e.g., an immunoglobulin, T cell receptor, HLA molecule, or chimeric antigen receptor). For example, an epitope can be a set of amino acid residues involved in recognition by a particular immunoglobulin; a Major Histocompatibility Complex (MHC) receptor; or in the context of T cells, those residues recognized by a T cell receptor protein and / or a chimeric antigen receptor. Epitopes can be prepared by isolation from a natural source, or they can be synthesized according to standard protocols in the art. Synthetic epitopes can comprise artificialamino acid residues, amino acid mimetics, (such as D isomers of naturally-occurring L amino acid residues or non-naturally-occurring amino acid residues). Throughout this disclosure, epitopes may be referred to in some cases as peptides or peptide epitopes. In certain embodiments, there is a limitation on the length of a peptide of the present disclosure. The embodiment that is length-limited occurs when the protein or peptide comprising an epitope described herein comprises a region (i.e., a contiguous series of amino acid residues) having 100% sequence identity with a native sequence. In order to avoid the definition of epitope from reading, e.g., on whole natural molecules, there is a limitation on the length of any region that has 100% sequence identity with a native peptide sequence. Thus, for a peptide comprising an epitope described herein and a region with 100% sequence identity with a native peptide sequence, the region with 100% sequence identity to a native sequence generally has a length of: less than or equal to 600 amino acid residues, less than or equal to 500 amino acid residues, less than or equal to 400 amino acid residues, less than or equal to 250 amino acid residues, less than or equal to 100 amino acid residues, less than or equal to 85 amino acid residues, less than or equal to 75 amino acid residues, less than or equal to 65 amino acid residues, and less than or equal to 50 amino acid residues. In certain embodiments, an “epitope” described herein is comprised by a peptide having a region with less than 51 amino acid residues that has 100% sequence identity to a native peptide sequence, in any increment down to 5 amino acid residues; for example 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues.

[0414] A “T cell epitope” refers to a peptide sequence bound by an MHC molecule in the form of a peptide-MHC (pMHC) complex. A peptide-MHC complex can be recognized and bound by a TCR of a T cell (e.g., a cytotoxic T-lymphocyte or a T-helper cell).

[0415] A “T cell” includes CD4+T cells and CD8+T cells. The term T cell also includes both T helper 1 type T cells and T helper 2 type T cells. T cells may be generated by the method described in the application, for a clinical application. T cells or adoptive T cells referred to here, such as for a clinical application are cells isolated from a biological source, manipulated and cultured ex vivo and prepared into a drug candidate for a specific therapy such as a cancer, e.g., melanoma. When drug candidate cells pass specific qualitative and quantitative criteria for fitness for a clinical application, the drug candidate may be designated a drug product. In some cases, a drug product is selected from a number of drug candidates. In the context of this application, a drug product is a T cell, more specifically, a population of T cells, or morespecifically a population of T cells with heterogeneous characteristics and subtypes. For example, a drug product, as disclosed herein may have a population of T cells comprising CD8+ T cells, CD4+ T cells, with cells at least above a certain exhibiting antigen specificity, a certain percentage of each exhibiting a memory phenotype, among others.

[0416] An “immune cell” refers to a cell that plays a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0417] An “immunogenic” peptide or an “immunogenic” epitope or an “immunogenic” peptide epitope is a peptide that binds to an HLA molecule and induces a cell-mediated or humoral response, for example, a cytotoxic T lymphocyte (CTL) response, a helper T lymphocyte (HTL) response and / or a B lymphocyte response. Immunogenic peptides described herein are capable of binding to an HLA molecule and thereafter induce a cell-mediated or humoral response (e.g., a CTL (cytotoxic) response, or a HTL response) to the peptide.

[0418] A “protective immune response” or “therapeutic immune response” refers to a CTL and / or an HTL response to an antigen derived from a pathogenic antigen (e.g., a tumor antigen), which in some way prevents or at least partially arrests disease symptoms, side effects or progression. The immune response can also include an antibody response which has been facilitated by the stimulation of helper T cells.

[0419] A “T cell receptor” (“TCR”) refers to a molecule, whether natural or partly or wholly synthetically produced, found on the surface of T lymphocytes (T cells) that recognizes an antigen bound to a major histocompatibility complex (MHC) molecule. The ability of a T cells to recognize an antigen associated with various diseases (e.g., cancers) or infectious organisms is conferred by its TCR, which is made up of both an alpha (a) chain and a beta (0) chain or a gamma (y) and a delta (8) chain. The proteins which make up these chains are encoded by DNA, which employs a unique mechanism for generating the tremendous diversity of the TCR. This multi-subunit immune recognition receptor associates with the CD3 complex and binds peptides presented by the MHC class I and II proteins on the surface of antigen-presenting cells (APCs). Binding of a TCR to a peptide on an APC is a central event in T cell activation.

[0420] As used herein, a “chimeric antigen receptor” or “CAR” refers to an antigen binding protein in that includes an immunoglobulin antigen binding domain (e.g., an immunoglobulin variable domain) and a T cell receptor (TCR) constant domain. As used herein, a “constant domain” of a TCR polypeptide includes a membrane-proximal TCR constant domain, a TCRtransmembrane domain and / or a TCR cytoplasmic domain, or fragments thereof. For example, in some embodiments, a CAR is a monomer that includes a polypeptide comprising an immunoglobulin heavy chain variable domain linked to a TCR0 constant domain. In some embodiments, the CAR is a dimer that includes a first polypeptide comprising an immunoglobulin heavy or light chain variable domain linked to a TCRa or TCR0constant domain and a second polypeptide comprising an immunoglobulin heavy or light chain variable domain (e.g., a K or A. variable domain) linked to a TCR0 or TCRa constant domain.

[0421] “Major Histocompatibility Complex” or “MHC” is a cluster of genes that plays a role in control of the cellular interactions responsible for physiologic immune responses. The terms “major histocompatibility complex” and the abbreviation “MHC” can include any class of MHC molecule, such as MHC class I and MHC class II molecules, and relate to a complex of genes which occurs in all vertebrates. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. Thus, a “Human Leukocyte Antigen” or “HLA” refers to a human Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., Immunology, 8THEd., Lange Publishing, Los Altos, Calif. (1994). For a detailed description of the MHC and HLA complexes, see, Paul, Fundamental Immunology, 3rdEd., Raven Press, New York (1993).

[0422] The major histocompatibility complex in the genome comprises the genetic region whose gene products expressed on the cell surface are important for binding and presenting endogenous and / or foreign antigens and thus for regulating immunological processes. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions. MHC proteins or molecules bind peptides and present them for recognition by T-cell receptors. The proteins encoded by the MHC can be expressed on the surface of cells, and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to a T-cell. MHC binding peptides can result from the proteolytic cleavage of protein antigens and represent potential lymphocyte epitopes, (e.g., T cell epitope and B cell epitope). MHCs can transport the peptides to the cell surface and present them there to specific cells, such as cytotoxic T-lymphocytes, T-helper cells, or B cells. The MHC region can be divided into three subgroups, class I, class II, and class III. MHC class I proteins can contain an a-chain and 02- microglobulin (not part of the MHC encoded by chromosome 15). They can present antigen fragments to cytotoxic T-cells. MHC class II proteins can contain a- and 0-chains and they can present antigen fragments to T-helper cells. MHC class III region can encode for other immunecomponents, such as complement components and cytokines. The MHC can be both polygenic (there are several MHC class I and MHC class II genes) and polymorphic (there are multiple alleles of each gene).

[0423] A “receptor” refers to a biological molecule or a molecule grouping capable of binding a ligand. A receptor may serve, to transmit information in a cell, a cell formation or an organism. A receptor comprises at least one receptor unit, for example, where each receptor unit may consist of a protein molecule. A receptor has a structure which complements that of a ligand and may complex the ligand as a binding partner. The information is transmitted in particular by conformational changes of the receptor following complexation of the ligand on the surface of a cell. In some embodiments, a receptor is to be understood as meaning in particular proteins of MHC classes I and II capable of forming a receptor / ligand complex with a ligand, in particular a peptide or peptide fragment of suitable length. A “ligand” refers to a molecule which has a structure complementary to that of a receptor and is capable of forming a complex with this receptor. In some embodiments, a ligand is to be understood as meaning a peptide or peptide fragment which has a suitable length and suitable binding motifs in its amino acid sequence, so that the peptide or peptide fragment is capable of forming a complex with MHC proteins such as MHC class I or MHC class II proteins. In some embodiments, a “receptor / ligand complex” is also to be understood as meaning a “receptor / peptide complex” or “receptor / peptide fragment complex”, including a peptide- or peptide fragment-presenting MHC molecule such as MHC class I or MHC class II molecules.

[0424] A “native” or a “wild type” sequence refers to a sequence found in nature. The term “naturally occurring” as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

[0425] The terms “peptide” and “peptide epitope” are used interchangeably with “oligopeptide” in the present specification to designate a series of residues connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acid residues. A “synthetic peptide” refers to a peptide that is obtained from a nonnatural source, e.g., is man-made. Such peptides can be produced using such methods as chemical synthesis or recombinant DNA technology. “Synthetic peptides” include “fusion proteins.”

[0426] The term “motif’ refers to a pattern of residues in an amino acid sequence of defined length, for example, a peptide of less than about 15 amino acid residues in length, or less than about 13 amino acid residues in length, for example, from about 8 to about 13 amino acid residues (e.g., 8, 9, 10, 11, 12, or 13) for a class I HLA motif and from about 6 to about 25 amino acid residues (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) for a class II HLA motif, which is recognized by a particular HLA molecule. Motifs are typically different for each HLA protein encoded by a given human HLA allele. These motifs differ in their pattern of the primary and secondary anchor residues. In some embodiments, an MHC class I motif identifies a peptide of 7, 8 9, 10, 11, 12 or 13 amino acid residues in length. In some embodiments, an MHC class II motif identifies a peptide of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 amino acid residues in length. A “cross-reactive binding” peptide refers to a peptide that binds to more than one member of a class of a binding pair member (e.g., a peptide bound by both a class I HLA molecule and a class II HLA molecule).

[0427] The term “residue” refers to an amino acid residue or amino acid mimetic residue incorporated into a peptide or protein by an amide bond or amide bond mimetic, or that is encoded by a nucleic acid (DNA or RNA). The nomenclature used to describe peptides or proteins follows the conventional practice. The amino group is presented to the left (the amino- or N-terminus) and the carboxyl group to the right (the carboxy- or C-terminus) of each amino acid residue. When amino acid residue positions are referred to in a peptide epitope, they are numbered in an amino to carboxyl direction with the first position being the residue located at the amino terminal end of the epitope, or the peptide or protein of which it can be a part. In the formulae representing selected specific embodiments of the present disclosure, the amino- and carboxyl-terminal groups, although not specifically shown, are in the form they can assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by standard three letter or single letter designations. The L- form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-form for those amino acid residues having D-forms is represented by a lower-case single letter or a lower case three letter symbol. However, when three letter symbols or full names are used without capitals, they can refer to L amino acid residues. Glycine has no asymmetric carbon atom and is simply referred to as “Gly” or “G”. The amino acid sequences of peptides set forth herein are generally designated using the standard single letter symbol. (A, Alanine; C, Cysteine; D, Aspartic Acid; E, Glutamic Acid; F, Phenylalanine; G, Glycine; H, Histidine; I, Isoleucine; K, Lysine; L, Leucine; M,Methionine; N, Asparagine; P, Proline; Q, Glutamine; R, Arginine; S, Serine; T, Threonine; V, Valine; W, Tryptophan; and Y, Tyrosine.)

[0428] A “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. 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). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate peptide function are well-known in the art.

[0429] “Pharmaceutically acceptable” refers to a generally non-toxic, inert, and / or physiologically compatible composition or component of a composition. A “pharmaceutical excipient” or “excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like. A “pharmaceutical excipient” is an excipient which is pharmaceutically acceptable.

[0430] According to the present disclosure, the term “vaccine” relates to a pharmaceutical preparation (pharmaceutical composition) or product that upon administration induces an immune response, for example, a cellular or humoral immune response, which recognizes and attacks a pathogen or a diseased cell such as a cancer cell. A vaccine may be used for the prevention or treatment of a disease. The term “individualized cancer vaccine” or “personalized cancer vaccine” “personal cancer vaccine” concerns a particular cancer patient and means that a cancer vaccine is adapted to the needs or special circumstances of an individual cancer patient.

[0431] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA, for example, mRNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. In some embodiments, the polynucleotide and nucleic acid can be in vitro transcribed mRNA. In some embodiments, the polynucleotide that is administered using the methods of the present disclosure is mRNA.

[0432] The terms “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides described herein do not contain some or all of the materials normally associated with the peptides in their in situ environment. For example, an “isolated” epitope can be an epitope that does not include the whole sequence of the protein from which the epitope was derived. For example, a naturally-occurring polynucleotide or peptide present in a living animal is not isolated, but the same polynucleotide or peptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such a polynucleotide could be part of a vector, and / or such a polynucleotide or peptide could be part of a composition, and still be “isolated” in that such vector or composition is not part of its natural environment. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules described herein, and further include such molecules produced synthetically. In some embodiments, a polypeptide, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure. The term “substantially pure” as used herein refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0433] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof. In some embodiments, two nucleic acids or polypeptides described herein are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as an amino acid sequence of a peptide or a coding region of a nucleotide sequence.

[0434] The term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

[0435] The terms “effective amount” or “therapeutically effective amount” or “therapeutic effect” refer to an amount of a therapeutic effective to “treat” a disease or disorder in a subject or mammal. The therapeutically effective amount of a drug has a therapeutic effect and as such can prevent the development of a disease or disorder; slow down the development of a disease or disorder; slow down the progression of a disease or disorder; relieve to some extent one or more of the symptoms associated with a disease or disorder; reduce morbidity and mortality; improve quality of life; or a combination of such effects.

[0436] The terms “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder. In some cases, treating may refer to reducing, or ameliorating a disorder and / or symptoms associated therewith (e.g., a neoplasia or tumor or infectious agent or an autoimmune disease). “Treating” can refer to administration of the therapy to a subject after the onset, or suspected onset, of a disease (e.g., cancer or infection by an infectious agent or an autoimmune disease). “Treating” includes the concepts of “alleviating”, which refers to lessening the frequency of occurrence or recurrence, or the severity, of any symptoms or other ill effects related to the disease and / or the side effects associated with therapy. The term “treating” may also encompass the concept of “managing” which refers to reducing the severity of a disease or disorder in a patient, e.g., extending the life or prolonging the survivability of a patient with the disease, or delaying its recurrence, e.g., lengthening the period of remission in a patient who had suffered from the disease. It is appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

[0437] The terms “prevent” or “prevention” refer to prophylactic or preventative measures that slow down the development of a targeted pathologic condition or disorder. Thus, those inneed of prevention include those prone to have the disorder or those in whom the disorder is to be prevented.

[0438] The term “depleted” when used to describe a cell sample (e.g., a peripheral blood mononuclear cell (PBMC) sample) refers to a cell sample in which a subpopulation of cells has been removed or depleted. For example, an immune cell sample depleted of CD25 expressing cells refers to an immune cell sample in which CD25 expressing cells have been removed or depleted. For example, one or more binding agents can be used to remove or deplete one or more cells or cell types from a sample. For example, CD14+cells can be depleted or removed from a PBMC sample, such as by using an antibody that binds to CD14.

[0439] The “stimulation” refers to a response induced by binding of a stimulatory molecule with its cognate ligand thereby mediating a signal transduction event. For example, stimulation of a T cell can refer to binding of a TCR of a T cell to a peptide-MHC complex. For example, stimulation of a T cell can refer to a step within protocol 1 or protocol 2 in which PBMCs are cultured together with peptide loaded APCs.

[0440] The term “enriched” refers to a composition or fraction wherein an object species has been partially purified such that the concentration of the object species is substantially higher than the naturally occurring level of the species in a finished product without enrichment. The term “induced cell” refers to a cell that has been treated with an inducing compound, cell, or population of cells that affects the cell’s protein expression, gene expression, differentiation status, shape, morphology, viability, and the like.

[0441] A “reference” can be used to correlate and / or compare the results obtained in the methods of the present disclosure from a diseased specimen. Typically, a “reference” may be obtained on the basis of one or more normal specimens, in particular specimens which are not affected by a disease, either obtained from an individual or one or more different individuals (e.g., healthy individuals), such as individuals of the same species. A “reference” can be determined empirically by testing a sufficiently large number of normal specimens.

[0442] As used herein, a tumor unless otherwise mentioned, is a cancerous tumor, and the terms cancer and tumor are used interchangeably throughout the document. While a tumor is a cancer of solid tissue, several of the compositions and methods described herein are in principle applicable to cancers of the blood, leukemia.Overview

[0443] T cell products have tremendous potential as therapeutic products, but there are significant challenges to developing profitable products that demonstrate efficacy, such assafe and robust genome engineering, in vivo persistence, and ex vivo cellular expansion. One important feature of a T cell product can be its sensitivity to peptide-HLA (pHLA) complexes as antigens - these complexes present diverse peptide epitopes to the extracellular environment that approximately represent the intracellular proteome. T cells can achieve this specificity via the T cell receptor (TCR), a heterodimeric protein complex that directly binds pHLA complexes and triggers downstream intracellular signaling pathways that culminate in an immune response (such as cytokine release or cytotoxicity). The present disclosure can preserve pHLA specificity of a TCR in a soluble format and can redirect a CAR-T cell product to pHLA-expressing target cells. These sTCR modules can constitute a universal modular system for multi-pHLA targeting that relies on the manufacturing of a single CAR-T cell product.

[0444] The present disclosure describes, inter alia, a technological and therapeutic approach whereby a soluble TCR (sTCR) protein, specific for particular pHLA antigen, may be fused to an epitope tag (e.g., the ALFA tag) and co-administered with a CAR-T cell product specific for the epitope tag (e.g., an anti-ALFA CAR-T cell). In some embodiments, the epitope tag is an ALFA-tag. The ALFA-tag may be a small, monomeric, well-soluble, and hydrophilic epitope tag, with balanced charges or uncharged, which may allow the ALFA-tag to have minimal impact on the physiological function of the protein the ALFA-tag is fused to. In some embodiments, the ALFA-tag can be devoid of residues prone to be modified by amine-reactive fixatives and cross-linkers. Thus, the ALFA-tag may not comprise any residues that are targets for aldehyde-based fixatives, which may allow it to be resistant to fixatives and be detectable even after fixation with solutions such as PFA or glutaraldehyde. In addition, the ALFA-tag sequence may be absent from common model organisms, which may allow reagents that bind to the ALFA-tag to have high specificity or may minimize the risk of cross-binding to native structures. The ALFA tag can comprise a sequence of SRLEEELRRRLTE (SEQ ID NO: 126).

[0445] The administered sTCR-ALFA protein module may decorate the anti-ALFA CAR-T cell via a specific and high affinity interaction between the ALFA tag and a VHH domain. After extracellular sTCR-ALFA module decoration, the CAR-T cells can become sensitive to the cognate pHLA antigen and can both productively bind pHLA multimers and may trigger intracellular pathways of T cell activation upon encounter with pHLA-expressing tumor cells (e.g., CD69 upregulation). Finally, these sTCR-ALFA decorated CAR-T cells can be capable of potent pHLA-specific tumor cytolysis.

[0446] In some cases, sTCRs with high affinity may interact with a target cell which has high cognate antigen density, which can lead to robust CAR-T activation and target cell killing. In some cases, sTCRs with low affinity may interact with a target cell which has a high cognate antigen density, which can lead to some CAR-T activation and some target cell killing. In some cases, sTCRs with high TCR affinity may interact with a target cell which has low cognate antigen density, which may not induce CAR-T activation or target cell killing. In some cases, sTCRs with low TCR affinity may interact with a target cell which has low cognate antigen density, which may not induce CAR-T activation or target cell killing.

[0447] In some cases, the proximal signaling strength of the CAR-T may be less than that of a non-CAR expressing T cell. In some cases, immune synapse formation by the CAR-T cell, sTCR, and the target cell may be deficient. In some cases, the sTCR / CAR-T complex may exhibit deficient rigidity.

[0448] To increase CAR-T activation, sTCRs may be engineered to increase proximal signaling and the CAR / sTCR may be designed to reduce immune synapse distance and flexibility. Other strategies may include designing other immunoreceptors which utilize native TCR-CD3 machinery or engineering T cells to express enhancers of T cell proximal signaling.

[0449] To enhance sensitivity, sTCRs may be designed to include two or more epitope tags. Inclusion of two or more epitope tags may further enhance the sensitivity of the sTCR- pHLA-CAR-T interaction by facilitating CAR pre-dimerization and proximal signaling. As described herein, the sTCR can be fused to two or more epitope tags. Without being bound by any particular theory, a sTCR fused to two or more epitope tags may engage multiple CAR molecules on the CAR-T cell simultaneously. This may result in increased T cell activation by crosslinking of CAR molecules and the amplification of the intracellular signaling pathways in the CAR-T cell.Soluble TCRs (sTCRs)

[0450] Provided herein is a composition comprising a T-cell receptor (TCR). In some embodiments, the TCR is a soluble TCR (sTCR). In some embodiments, the TCR has a general structure and backbone designed and optimized for action with placeholder antigenspecificity (e.g., CDRs of the alpha variable region and / or CDRs of the beta variable region) sequences, which may be termed a scaffold. In essence, a scaffold may be a soluble TCR design of the disclosure. In some embodiments, the TCR has at least one tag (tag-sTRCR). In some embodiments, the composition is a polynucleotide encoding the tag-sTCR. In someembodiments, the tag-sTCR binds to a peptide:MHC complex of a target cell. In some embodiments, the target cell is a cancer cell.

[0451] In some embodiments, the composition further comprises a chimeric antigen receptor (CAR). In some embodiments, the composition further comprises a polynucleotide encoding chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an extracellular domain comprising an anti-tag binding domain. In some embodiments, the anti-tag binding domain binds to the at least one tag of the tag-sTCR. In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the CAR comprises an intracellular domain. In some embodiments, the CAR is expressed on the surface of a cell. In some embodiments the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD4 T cell. In some embodiments, the T cell is a CD8 T cell.

[0452] Also provided herein is a composition comprising a tag-sTCR and an immune cell. In some embodiments, the TCR is a soluble TCR (sTCR). In some embodiments, the TCR has at least one tag (tag-sTRCR). In some embodiments, the composition comprises a polynucleotide encoding the tag-sTCR. In some embodiments, the immune cell comprises a CAR. In some embodiments, the CAR comprises an extracellular domain comprising an antitag binding domain. In some embodiments, the anti-tag binding domain binds to the at least one tag of the tag-sTCR. In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the CAR comprises an intracellular domain. In some embodiments, the CAR is expressed on the surface of a cell. In some embodiments the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD4 T cell. In some embodiments, the T cell is a CD8 T cell. In some embodiments, the tag-sTCR binds to a peptide:MHC complex of a target cell. In some embodiments, the target cell is a cancer cell.

[0453] In some embodiments, the soluble TCR comprises a peptide:MHC complex engager. In some embodiments, the peptide:MHC complex engager comprises a TCR alpha variable domain. In some embodiments, the peptide:MHC complex engager comprises a TCR beta variable domain. In some embodiments, the peptide:MHC complex engager comprises a TCR alpha variable domain and a TCR beta variable domain.

[0454] In some embodiments, the soluble TCR is not a single chain TCR. In some embodiments, the soluble TCR does not comprise a constant region of a TCR.

[0455] In some embodiments, the soluble TCR is a single chain TCR. In some embodiments, the TCR alpha variable domain is operably linked to the TCR beta variable domain. In some embodiments, the soluble TCR comprises, form N-terminus to C-terminus, the TCR alpha variable domain operably linked to the TCR beta variable domain. In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the TCR beta variable domain operably linked to the TCR alpha variable domain.

[0456] In some embodiments, the tag-sTCR is a fusion protein. In some embodiments, the at least one tag is operably linked to the soluble TCR. In some embodiments, the at least one tag is operably linked to the N-terminus of the soluble TCR. In some embodiments, the at least one tag is operably linked to the C-terminus of the soluble TCR.

[0457] In one aspect, a recombinant nucleic acid encoding the sTCR scaffold may comprise a sequence encoding a single polypeptide. The single polypeptide can comprise a TRAV domain, wherein the TRAV framework sequences are optimized, and the CDR1, CDR2, and CDR3 are changeable based on the target antigen, wherein the TRAV is operably linked with a sequence encoding a TRAC - which is in turn connected via a sequence encoding a post- translational auto-cleavage sequence, for example P2A, to a sequence encoding a polypeptide comprising the TRBC that is operably linked to a TRBV, wherein the TRBV framework sequences are optimized, and the CDR1, CDR2, and CDR3 are changeable based on the target antigen.

[0458] In some embodiments, the soluble TCR scaffold comprises a human TRAC engineered to comprise one or more stability enhancing mutations. In some embodiments, the soluble TCR scaffold comprises a human TRBC engineered to comprise one or more stability enhancing mutations. In some embodiments, the soluble TCR scaffold comprises a human TRAC engineered to comprise one or more of stability enhancing mutations: S139F, T150I, A190T and the TRBC comprises E134K, H139R, D155P, S170D.

[0459] In some embodiments, the scaffold comprises a human TRAC engineered to comprise one or more of stability enhancing mutations: S139F, T150I, or A190T, and a human TRBC engineered to comprise one or more of stability enhancing mutations: S170D, C191A or N205D. In some embodiments, both TRAC and TRBC sequences may comprise one or more N>Q substitutions. The numbering of the amino acid residues in the TRAC and TRBC domains used herein follows the Kabat numbering system (Kabat, et al. Sequences of Immunological Interest Vol. 1 Fifth Edition 1991 US Department of Health and Human Services, Public Health Service, NIH). In some embodiments, the single chain soluble TCRdescribed herein does not comprise any stability enhancing mutations in the TRAC and TRBC domains.

[0460] In some embodiments, the soluble TCR scaffold comprises a human TRAV engineered to comprise one or more stability enhancing mutations. In some embodiments, the soluble TCR scaffold comprises a human TRBV engineered to comprise one or more stability enhancing mutations. In some embodiments, the TRAV is engineered to include stability enhancing mutation comprises X96L, wherein X represents any amino acid at position 96 of the TCR alpha variable domain as determined by IMGT numbering scheme. In some embodiments, the TRBV is engineered to include stability enhancing mutation comprising one or more of X9R and XI 0Y, wherein X represents any amino acid at position 9 or 10 of the TCR beta variable domain as determined by IMGT numbering scheme.

[0461] In some embodiments, the soluble TCR is a single chain TCR. In some embodiments, the single chain TCR comprises, from N-terminus to C-terminus, the TCR beta variable domain, a TCR beta constant domain, the TCR alpha variable domain, and a TCR alpha constant domain. In some embodiments, the soluble TCR comprises, from N-terminus to C- terminus, the TCR alpha variable domain, a TCR alpha constant domain, the TCR beta variable domain, and a TCR beta constant domain. In some embodiments, the at least one tag is operably linked to the N-terminus of the soluble TCR. In some embodiments, the at least one tag is operably linked to the TCR beta variable domain. In some embodiments, the at least one tag is an ALFA-tag. In some embodiments, the soluble TCR comprises a first linker. In some embodiments, the at least one tag is operably linked to the TCR beta variable domain via the first linker. In some embodiments, the first linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10. In some embodiments, the n is 1. In some embodiments, the soluble TCR comprises a second linker. In some embodiments, the second linker is operably linked to the TCR beta constant domain and the TCR alpha variable domain. In some embodiments, the TCR alpha constant domain is operably linked to the at least one tag via the second linker. In some embodiments, the second linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10. In some embodiments, n is 7. In some embodiments, n is 4. In some embodiments, the soluble TCR comprises an additional tag operably linked to the C-terminus of the soluble TCR. In some embodiments, the additional tag is a lOXHIS-sortag operably linked to the C-terminus of the soluble TCR. In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the at least one tag, the TCR beta variable domain, the TCR beta constant domain, the secondlinker, the TCR alpha variable domain, the TCR alpha constant domain, and the additional tag. In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the at least one tag, the first linker, the TCR beta variable domain, the TCR beta constant domain, the second linker, the TCR alpha variable domain, the TCR alpha constant domain, and the additional tag. In some embodiments, the soluble TCR comprises, from N-terminus to C- terminus, the TCR alpha variable domain, the TCR alpha constant domain, the second linker, the at least one tag, the TCR beta variable region, the TCR beta constant region, and the additional tag. In some embodiments, the soluble TCR comprises, from N-terminus to C- terminus, the TCR alpha variable domain, the TCR alpha constant domain, the second linker, the at least one tag, the first linker, the TCR beta variable region, the TCR beta constant region, and the additional tag.

[0462] In some embodiments, the single chain TCR comprises one or more disulfide bridges between the TCR alpha constant domain and the TCR beta constant domain. In some embodiments, the single chain TCR comprises a modification comprising removal of one or more N-glycosylation sites in the TCR alpha constant domain and / or TCR beta constant domain. In some embodiments, removal of the one or more N-glycosylation sites comprises substituting one or more asparagine residue to one or more aspartic acid residues or one or more glutamic acid residues. In some embodiments, single chain TCR comprises a modification comprising removal of one or more unpaired cysteines in the TCR alpha constant domain and / or TCR beta constant domain. In some embodiments, removal of the one or more unpaired cysteines comprises substituting one or more unpaired cysteines to one or more serine residues. In some embodiments, single chain TCR comprises one or more disulfide bonds in the TCR alpha constant domain and / or the TCR beta constant domain. In some embodiments, the single chain TCR comprises one or more mutations in the TCR alpha constant domain and / or the TCR beta constant domain. In some embodiments, the one or more mutations are one or more stability enhancing mutations. In some embodiments, the one or more mutations in the TCR alpha constant domain comprises S139F, T150I, or A190T. In some embodiments, the one or more mutations in the TCR beta constant domain comprises C191A, N205D, E134K, H139R, D155P, or S170D. In some embodiments, TCR alpha constant domain and the TCR beta constant domain comprises one or more N>Q substitutions. In some embodiments, the single chain TCR comprises no mutations in the TCR alpha constant domain and / or the TCR beta constant domain.

[0463] In some embodiments, the soluble TCR is a single chain TCR. In some embodiments, the TCR comprises, from N-terminus to C-terminus, the TCR beta variable domain, the TCR beta constant domain, the TCR alpha variable domain, and the TCR alpha constant domain. In some embodiments, the at least one tag is operably linked to the N-terminus of the soluble TCR. In some embodiments, the soluble TCR comprises an anti-CD3 ScFv region operably linked to the N-terminus of the soluble TCR. In some embodiments, the soluble TCR comprises a linker. In some embodiments, the linker is operably linked to the TCR alpha constant domain and the TCR alpha variable domain. In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10. In some embodiments, n is 7. In some embodiments, the soluble TCR is a single chain TCR. In some embodiments, the TCR comprises from N-terminus to C-terminus, the TCR alpha variable domain, the TCR alpha constant domain, the TCR beta variable domain, and the TCR beta constant domain. In some embodiments, the at least one tag is operably linked to the TCR beta variable domain. In some embodiments, the soluble TCR comprises an anti-CD3 ScFv region operably linked to the TCR beta variable domain. In some embodiments, the soluble TCR comprises a linker. In some embodiments, the linker is operably linked to the at least one tag or the anti-ScFv region and the TCR beta constant domain. In some embodiments, the linker is operably linked to the at least one tag and the TCR beta constant domain. In some embodiments, the linker is operably linked to the anti-ScFv region and the TCR beta constant domain. In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10. In some embodiments, n is 4. In some embodiments, the soluble TCR comprises an additional tag operably linked to the C-terminus of the soluble TCR. In some embodiments, the additional tag is a lOXHIS-sortag operably linked to the C-terminus of the soluble TCR. In some embodiments, the soluble TCR comprises a lOXHIS-sortag operably linked to the C-terminus of the soluble TCR. In some embodiments, the soluble TCR comprises one or more disulfide bridges between the TCR alpha constant domain and the TCR beta constant domain. In some embodiments, the soluble TCR comprises a modification comprising removal of one or more N-glycosylation sites in the TCR alpha constant domain and / or TCR beta constant domain. In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the TCR alpha variable domain, the TCR alpha constant domain, the linker, the at least one tag, the TCR beta variable region, the TCR beta constant region, and the lOXHIS-sortag. In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the at least one tag, the TCR beta variable domain, the TCR beta constantdomain, the linker, the TCR alpha variable domain, the TCR alpha constant domain, and the lOXHIS-sortag.

[0464] In some embodiments, the TCR alpha variable domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 421. In some embodiments, the TCR alpha variable domain comprises a sequence set forth in SEQ ID NO: 421. In some embodiments, the TCR alpha constant domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 492. In some embodiments, the TCR alpha constant domain comprises a sequence set forth in SEQ ID NO: 492. In some embodiments, the TCR alpha chain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 491. In some embodiments, the TCR alpha chain comprises a sequence set forth in SEQ ID NO: 491. In some embodiments, the TCR beta variable domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 427. In some embodiments, the TCR beta variable domain comprises a sequence set forth in SEQ ID NO: 427. In some embodiments, the TCR beta constant domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 490. In some embodiments, the TCR beta constant domain comprises a sequence set forth in SEQ ID NO: 490. In some embodiments, the TCR beta chain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 132. In some embodiments, the TCR beta chain comprises a sequence set forth in SEQ ID NO: 132. In some embodiments, the TCR comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 489. In some embodiments, the TCR comprises a sequence having a sequence set forth in SEQ ID NO: 489.

[0465] In some embodiments, the TCR alpha variable domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 421. In some embodiments, the TCR alpha variable domain comprises a sequence set forth in SEQ ID NO: 421. In some embodiments, the TCR alpha constant domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 492. Insome embodiments, the TCR alpha constant domain comprises a sequence set forth in SEQ ID NO: 492. In some embodiments, the TCR alpha chain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 496. In some embodiments, the TCR alpha chain comprises a sequence set forth in SEQ ID NO: 496. In some embodiments, the TCR beta variable domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 427. In some embodiments, the TCR beta variable domain comprises a sequence set forth in SEQ ID NO: 427. In some embodiments, the TCR beta constant domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 490. In some embodiments, the TCR beta constant domain comprises a sequence set forth in SEQ ID NO: 490. In some embodiments, the TCR beta chain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 495. In some embodiments, the TCR beta chain comprises a sequence set forth in SEQ ID NO: 495. In some embodiments, the TCR comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 494. In some embodiments, the TCR comprises a sequence having a sequence set forth in SEQ ID NO: 494.

[0466] In some embodiments, the TCR alpha variable domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 421. In some embodiments, the TCR alpha variable domain comprises a sequence set forth in SEQ ID NO: 421. In some embodiments, the TCR alpha constant domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 501. In some embodiments, the TCR alpha constant domain comprises a sequence set forth in SEQ ID NO: 501. In some embodiments, the TCR alpha chain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 500. In some embodiments, the TCR alpha chain comprises a sequence set forth in SEQ ID NO: 500. In some embodiments, the TCR beta variable domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 427. In some embodiments, the TCR beta variable domain comprises a sequence set forth in SEQ ID NO:427. In some embodiments, the TCR beta constant domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 499. In some embodiments, the TCR beta constant domain comprises a sequence set forth in SEQ ID NO: 499. In some embodiments, the TCR beta chain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 498. In some embodiments, the TCR beta chain comprises a sequence set forth in SEQ ID NO: 498. In some embodiments, the TCR comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence set forth in SEQ ID NO: 497. In some embodiments, the TCR comprises a sequence having a sequence set forth in SEQ ID NO: 497.

[0467] In some embodiments, the TCR alpha variable domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from the group consisting of the TCR alpha variable domain sequences of Tables 5, 6, 8, 11, and 12. In some embodiments, the TCR beta variable domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from the group consisting of the TCR beta variable domain sequences of Tables 5, 6, 8, 11, and 12. In some embodiments, the TCR alpha constant domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from the group consisting of the TCR alpha constant domain sequences of Tables 5, 6, 8, 11, and 12. In some embodiments, the TCR beta constant domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from the group consisting of the TCR beta constant domain sequences of Tables 5, 6, 8, 11, and 12. In some embodiments, wherein the additional tag comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% identify to a sequence of SEQ ID NO: 131. In some embodiments, wherein the additional tag comprises a sequence of SEQ ID NO: 131.

[0468] In some embodiments, the single chain TCR disclosed herein exhibits anti-tumor cytotoxicity at a level comparable to a corresponding heterodimeric soluble TCR.

[0469] In some embodiments, the single chain TCR disclosed herein induces T cell proliferation at a level comparable to a corresponding heterodimeric soluble TCR.

[0470] In some embodiments, the single chain TCR disclosed herein upregulates expression of one or more cytotoxicity markers at a level comparable to a corresponding heterodimeric soluble TCR.

[0471] In some embodiments, the at least one tag binds to the anti-tag binding domain of the CAR. In some embodiments, the anti-tag binding domain is an antibody or antigen binding fragment thereof. In some embodiments, the at least one tag is not an affinity tag. In some embodiments, the at least one tag does not comprise a fluorophore. In some embodiments, the tag-sTCR does not comprise a scFv. In some embodiments, the at least one tag is a polypeptide that is absent in eukaryotic systems.

[0472] In some embodiments, the at least one tag comprises less than 500, less than 450, less than 400, less than 350 , less than 300, less than 250 , less than 200, less than 150, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, less than 10, or less than 5 amino acids. In some embodiments, the at least one tag comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, or at least 300 or more amino acids.

[0473] In some embodiments, the at least one tag does not comprise modified amino acids. In some embodiments, the at least one tag does not comprise unnatural amino acids. In some embodiments, the at least one tag does not comprise modified amino acids or unnatural amino acids. In some embodiments, all amino acids of the at least one tag are non-modified amino acids with natural peptide bonds. In some embodiments, the at least one tag forms a stable alpha-helical structure. In some embodiments, the at least one tag is hydrophilic. In some embodiments the at least one tag is neutral at physiological pH. In some embodiments, the at least one tag is hydrophilic and is neutral at physiological pH. In some embodiments, the at least one tag comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 126. In some embodiments, the at least one tag comprises a sequence of SEQ ID NO: 126. In some embodiments, the at least one tag comprises a sequence that comprises less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 amino acids differing from SEQ ID NO: 126. In some embodiments, the at least one tag further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more amino acids on the N-terminus of the sequence. In some embodiments, the at least one tag further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more aminoacids on the C-terminus of the sequence. In some embodiments, the at least one tag further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more amino acids on both the N-terminus and C- terminus of the sequence.

[0474] In some embodiments, the at least one tag is recognized by the anti-tag binding domain of the CAR with a Ka of less than 3,000 nM, less than 2,500 nM, less than 2,000 nM, less than 1,500 nM, less than 1,000 nM, less than 500 nM, less than 250 nM, less than 150 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, or less than 1 nM. In some embodiments, the at least one tag is recognized by the anti-tag binding domain of the CAR with a Ka of about 1 nM to about 10 nM, about 10 nM to about 20 nM, about 20 nM to about 30 nM, about 30 nM to about 40 nM, about 40 nM to about 50 nM, about 50 nM to about 60 nM, about 60 nM to about 70 nM, about 70 nM to about 80 nM, about 80 nM to about 90 nM, about 90 nM to about 100 nM, about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, or about 900 nM to about 1000 nM. In some embodiments, the peptide:MHC complex engager specifically binds to an endogenous disease specific antigen. In some embodiments, the peptide:MHC complex engager specifically binds to a cancer antigen. In some embodiments, the cancer antigen is a neoantigen. In some embodiments, the cancer antigen is a tumor associated antigen. In some embodiments, the cancer antigen is a viral antigen. In some embodiments, the peptide:MHC complex engager binds to the peptide:MHC complex comprising an epitope from the endogenous disease specific antigen that is presented on the surface of the target cell. In some embodiments, the target cell is a cancer cell.

[0475] In some embodiments, the peptide:MHC complex engager binds to the peptide:MHC complex with a Ka of less than 3,000 nM, less than 2,500 nM, less than 2,000 nM, less than 1,500 nM, less than 1,000 nM, less than 500 nM, less than 250 nM, less than 150 nM, less than 100 nM, less than 90 nM, 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, or less than 1 nM. In some embodiments, the peptide:MHC complex engager binds to the peptide:MHC complex with a Ka of about 1 nM to about 10 nM, about 10 nM to about 20 nM, about 20 nM to about 30 nM, about 30 nM to about 40 nM, about 40 nM to about 50 nM, about 50 nM to about 60 nM, about 60 nM to about 70 nM, about 70 nM to about 80 nM, about 80 nM to about 90 nM, about 90 nM toabout 100 nM, about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, or about 900 nM to about 1000 nM. In some embodiments, the MHC of the peptide:MHC complex is a class I MHC. In some embodiments, the MHC of the peptide:MHC complex is a class II MHC.

[0476] In some embodiments, the epitope is derived from a RAS polypeptide or a fragment thereof. In some embodiments, the RAS polypeptide is a mutated RAS polypeptide. In some embodiments, the epitope derived from the RAS polypeptide or a fragment thereof comprises a G12C mutation. In some embodiments, the epitope derived from the RAS polypeptide or a fragment thereof comprises a G12V mutation. In some embodiments, the epitope derived from the RAS polypeptide or a fragment thereof comprises a G12D mutation. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 199. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 199. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 200. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 200. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 201. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 201. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 202. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 202. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 203. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 203. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 204. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 204. In someembodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 205. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 205. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 359. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 359. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 432. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 432. In some embodiments, an MHC of the peptide:MHC complex comprising the epitope derived from the RAS polypeptide or a fragment thereof comprises an MHC encoded by an HLA-A*02:01 allele. In some embodiments, an MHC of the peptide:MHC complex comprising the epitope derived from the RAS polypeptide or a fragment thereof comprises an MHC encoded by an HLA-C*08:02 allele.

[0477] In some embodiments, the epitope is derived from a GATA3 polypeptide or a fragment thereof. In some embodiments, the GATA3 polypeptide is a mutated GATA3 polypeptide. In some embodiments, the GATA3 polypeptide is encoded by a neoORF. In some embodiments, the epitope derived from a GAT A3 polypeptide or a fragment thereof has a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the epitope has a sequence of SEQ ID NO: 15.

[0478] In some embodiments, the epitope is derived from a Human Papillomavirus (HPV) protein. In some embodiments, the HPV protein is a HPV16 E7 protein. In some embodiments, the epitope has a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 458. In some embodiments, the epitope has a sequence of SEQ ID NO: 458.

[0479] In some embodiments, the single chain TCR comprises a linker. In some embodiments, the linker is between the TCR alpha variable domain and the TCR beta variable domain. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker comprises the at least one tag sequence. In some embodiments, the linker comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 125. In some embodiments, the linker has asequence of SEQ ID NO: 125. In some embodiments, the linker comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 128. In some embodiments, the linker has a sequence of SEQ ID NO: 128. In some embodiments, the linker comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 135. In some embodiments, the linker has a sequence of SEQ ID NO: 135. In some embodiments, the linker comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 488. In some embodiments, the linker has a sequence of SEQ ID NO: 488.

[0480] In some embodiments, the soluble TCR is a single chain TCR, and wherein the single chain TCR comprises, from N terminus to C terminus, the TCR alpha variable domain, the TCR alpha constant domain, a first linker, a first tag, a second linker, a second tag, the TCR beta variable domain, and the TCR beta constant domain. In some embodiments, the first tag or the second tag comprises a sequence of SEQ ID NO: 126. In some embodiments, the first tag and the second tag each comprises a sequence of SEQ ID NO: 126. In some embodiments, the first linker or the second linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10. In some embodiments, the integer is 1. In some embodiments, the integer is 2. In some embodiments, the integer is 3. In some embodiments, the integer is 4. In some embodiments, the integer is 5. In some embodiments, the integer is 6. In some embodiments, the integer is 7. In some embodiments, the integer is 8. In some embodiments, the integer is 9. In some embodiments, the integer is 10. In some embodiments, the first linker comprises a sequence of (GGGGS)4, and the second linker comprises a sequence of GGGGS. In some embodiments, the first linker comprises a sequence of (GGGGS)2, and the second linker comprises a sequence of GGGGS.

[0481] In some embodiments, the soluble TCR comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 552, 556, and 557. In some embodiments, the soluble TCR comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO: 552. In some embodiments, the soluble TCR comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO: 556. In some embodiments, the soluble TCR comprises a sequence having at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO: 557.

[0482] In some embodiments, the TCR alpha variable domain and the TCR beta variable domain are on two separate polypeptide chains comprising a first polypeptide chain and a second polypeptide chain. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 138, 541, 543, 544, 545, 547, 548, and 550. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 138. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 541. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 543. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 544. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 545. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 547. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 548. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 550.

[0483] In some embodiments, the first polypeptide chain comprises a first dimerizing domain. In some embodiments, the first dimerizing domain is fused to the TCR alpha variable domain. In some embodiments, the second polypeptide chain comprises a second dimerizing domain. In some embodiments, the second dimerizing domain is fused to the TCR beta variable domain. In some embodiments, the first dimerizing domain comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25 or more amino acids of a TCR extracellular domain adjacent to a TCR transmembrane domain. In some embodiments, the second dimerizing domain comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25 or more amino acids of a TCR extracellular domain adjacent to a TCR transmembrane domain.

[0484] In some embodiments, the first dimerizing domain comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 425. In some embodiments, the first dimerizing domain has a sequence of SEQ ID NO: 425. In some embodiments, the second dimerizing domain comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 431. In some embodiments, the second dimerizing domain has a sequence of SEQ ID NO: 431.

[0485] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain and the first dimerizing domain; and the second polypeptide comprises, from N terminus to C terminus, the TCR beta variable domain and the second dimerizing domain. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, a first peptide linker, and the first dimerizing domain; and the second polypeptide comprises, from N terminus to C terminus, the TCR beta variable domain, a second peptide linker, and the second dimerizing domain. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain and the TCR alpha variable domain, and the second polypeptide comprises, from N terminus to C terminus, the second dimerizing domain and the TCR beta variable domain. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain, a first peptide linker, and the TCR alpha variable domain, and the second polypeptide comprises, from N terminus to C terminus, the second dimerizing domain, a second peptide linker, and the TCR beta variable domain.

[0486] In some embodiments, the first dimerizing domain comprises a TCR alpha constant (TRAC) domain or portion thereof. In some embodiments, the second dimerizing domain comprises a TCR beta constant (TRBC) domain or portion thereof. In some embodiments, the first dimerizing domain comprises a TCR alpha constant (TRAC) domain or portion thereof and the second dimerizing domain comprises a TCR beta constant (TRBC) domain or portion thereof.

[0487] In some embodiments, the first dimerizing domain and the second dimerizing domain are linked. In some embodiments, the first dimerizing domain and the second dimerizing domain are linked by a single disulfide bridge. In some embodiments, the first dimerizing domain and the second dimerizing domain are linked by two or more disulfide bridges.

[0488] In some embodiments, the first polypeptide chain comprises the at least one tag. In some embodiments, the first polypeptide chain comprises from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, a linker, and the at least one tag. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the TCR alpha variable domain, and the first dimerizing domain. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, a linker, the TCR alpha variable domain, and the first dimerizing domain. In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10 or more. In some embodiments, the integer is from 1 to 10. In some embodiments, the integer is 1. In some embodiments, the integer is 2. In some embodiments, the integer is 3. In some embodiments, the integer is 4. In some embodiments, the integer is 5. In some embodiments, the integer is 6. In some embodiments, the integer is 7. In some embodiments, the integer is 8. In some embodiments, the integer is 9. In some embodiments, the integer is 10.

[0489] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain, the TCR alpha variable domain, and the at least one tag. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the first dimerizing domain, and the TCR alpha variable domain.

[0490] In some embodiments, the second polypeptide chain comprises the at least one tag. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the TCR beta variable domain, and the second dimerizing domain. In some embodiments, wherein the second polypeptide chain comprises, from N terminus to Cterminus, the at least one tag, a linker, the TCR beta variable domain, and the second dimerizing domain. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, and the at least one tag. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, a linker, and the at least one tag. In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10 or more. In some embodiments, the integer is from 1 to 10. In some embodiments, the integer is 1. In some embodiments, the integer is 2. In some embodiments, the integer is 3. In some embodiments, the integer is 4. In some embodiments, the integer is 5. In some embodiments, the integer is 6. In some embodiments, the integer is 7. In some embodiments, the integer is 8. In some embodiments, the integer is 9. In some embodiments, the integer is 10.

[0491] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the second dimerizing domain, the TCR alpha beta variable domain, and the at least one tag. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the second dimerizing domain, and the TCR beta variable domain.

[0492] In some embodiments, the at least one tag comprises two or more tags. In some embodiments, the at least one tag comprises two tags. In some embodiments, the at least one tag comprises three tags. In some embodiments, the at least one tag comprises four tags. In some embodiments, the at least one tag comprises five tags. In some embodiments, each of the two or more tags comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 126. In some embodiments, the at least one tag comprises a sequence of SEQ ID NO: 126. In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10 or more. In some embodiments, the integer is from 1 to 10. In some embodiments, the integer is 1. In some embodiments, the integer is 2. In some embodiments, the integer is 3. In some embodiments, the integer is 4. In some embodiments, the integer is 5. In some embodiments, the integer is 6. In some embodiments, the integer is 7. In some embodiments, the integer is 8. In some embodiments, the integer is 9. In some embodiments, the integer is 10.

[0493] In some embodiments, the at least one tag comprises two tags. In some embodiments a sTCR comprising two tags results in killing of target cells at a peptide concentration less than IxlO'6M, less than 5xl0'7M, less than IxlO'7M, less than 5xl0'8M, or less than IxlO'8M, less than 5xlO'9M, or less than IxlO'9M. In some embodiments a sTCR comprising two tags results in Ki-67 expression at a peptide concentration less than IxlO'6M, less than 5xl0'7M, less than IxlO'7M, less than 5xl0'8M, or less than IxlO'8M, less than 5xl0'9M, or less than IxlO'9M. In some embodiments a sTCR comprising two tags results in Granzyme B production at a peptide concentration less than IxlO'6M, less than 5xl0'7M, less than IxlO'7M, less than 5xl0'8M, or less than IxlO'8M, less than 5xl0'9M, or less than IxlO'9M. In some embodiments, a sTCR comprising two tags results in a 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or more increase in percent of target cells killed relative to an otherwise identical sTCR comprising one tag. In some embodiments, a sTCR comprising two tags results in a 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or more increase in Ki -67 expression relative to an otherwise identical sTCR comprising one tag. In some embodiments, a sTCR comprising two tags results in a 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or more increase in Granzyme B production relative to an otherwise identical sTCR comprising one tag.

[0494] In some embodiments, the at least one tag comprises three tags. In some embodiments a sTCR comprising three tags results in killing of target cells at a peptide concentration less than IxlO'6M, less than 5xl0'7M, less than IxlO'7M, less than 5xl0'8M, or less than IxlO'8M, less than 5xl0'9M, or less than IxlO'9M. In some embodiments a sTCR comprising three tags results in Ki-67 expression at a peptide concentration less than IxlO'6M, less than 5xl0'7M, less than IxlO'7M, less than 5xl0'8M, or less than IxlO'8M, less than 5xl0'9M, or less than IxlO'9M. In some embodiments a sTCR comprising three tags results in Granzyme B production at a peptide concentration less than IxlO'6M, less than 5xl0'7M, less than IxlO'7M, less than 5xl0'8M, or less than IxlO'8M, less than 5xl0'9M, or less than IxlO'9M. In some embodiments, a sTCR comprising three tags results in a 5- fold, 10-fold, 15-fold, 20-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or more increase in percent of target cells killed relative to an otherwise identical sTCR comprising two tags. In some embodiments, a sTCR comprising three tags results in a 5-fold, 10-fold, 15-fold, 20- fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or more increase in Ki-67 expression relative to an otherwise identical sTCR comprising two tags. In some embodiments, a sTCR comprising three tags results in a 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or more increase in Granzyme B production relative to an otherwise identical sTCR comprising two tags.

[0495] In some embodiments, the two or more tags are linked to the same first polypeptide chain. In some embodiments, the two or more tags are linked to the same second polypeptide chain. In some embodiments, the two or more tags are linked to different polypeptide chains.

[0496] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, and the two or more tags operably linked. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, a first linker, a first tag, a second linker, and a second tag. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the two or more tags operably linked, the TCR alpha variable domain, and the first dimerizing domain. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, a first tag, a first linker, a second tag, a second linker, the TCR alpha variable domain, and the first dimerizing domain. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, and the two or more tags operably linked. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, a first linker, a first tag, a second linker, and a second tag. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the two or more tags operably linked, the TCR beta variable domain, and the second dimerizing domain. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, a first tag, a first linker, a second tag, a second linker, the TCR beta variable domain, and the second dimerizing domain. In some embodiments, the first tag and the second tag comprise the same sequence. In some embodiments, the sequence comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 126. In some embodiments, the at least one tag comprises a sequence of SEQ ID NO: 126. In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10 or more. In some embodiments, the integer is from 1 to 10. In some embodiments, the integer is 1. In some embodiments, the integer is 2. In some embodiments, the integer is 3. In some embodiments, the integer is 4. In some embodiments, the integer is 5. In some embodiments, the integer is 6. In some embodiments, the integer is 7. In some embodiments, the integer is 8. In some embodiments, the integer is 9. In some embodiments, the integer is 10.

[0497] In some embodiments, the soluble TCR comprises two or more soluble TCRs. In some embodiments, each soluble TCR of the two or more soluble TCRs recognizes a different peptide:MHC complex. In some embodiments, the different peptide:MHC complexes are on the same target cell. In some embodiments, the different peptide:MHC complexes are on different target cells. In some embodiments, the different peptide:MHC complexes comprise different epitope sequences. In some embodiments, the different peptide:MHC complexes comprise different MHC molecules encoded by different HLA alleles. In some embodiments, the different peptide:MHC complexes comprise different epitope sequences presented by the same MHC molecule encoded by the same HLA allele. In some embodiments, the different peptide:MHC complexes comprise different epitope sequences presented by different MHC molecules encoded by different HLA alleles. In some embodiments, the different peptideMHC complexes comprise the same epitope sequence presented by different MHC molecule encoded by different HLA alleles.

[0498] In some embodiments, the soluble TCR comprises a TCR beta variable domain comprising a sequence comprising a complementarity determining region 3 (CDR3). In some embodiments, the CDR3 comprises an amino acid with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 430. In some embodiments, CDR3 comprises an amino acid sequence of SEQ ID NO: 430. In some embodiments, the CDR3 comprises an amino acid with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 435. In some embodiments, CDR3 comprises an amino acid sequence of SEQ ID NO: 435. In some embodiments, the CDR3 comprises an amino acid with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 445. In some embodiments, CDR3 comprises an amino acid sequence of SEQ ID NO: 445. In some embodiments, the CDR3 comprises an amino acid with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 456. In some embodiments, CDR3 comprises an amino acid sequence of SEQ ID NO: 456. In some embodiments, the TCR beta variable domain comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 327. In some embodiments, TCR beta variable domain comprises an amino acid sequence of SEQ ID NO: 327. In some embodiments, the TCR beta variable domain comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ IDNO: 434. In some embodiments, TCR beta variable domain comprises an amino acid sequence of SEQ ID NO: 434. In some embodiments, the TCR beta variable domain comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 442. In some embodiments, TCR beta variable domain comprises an amino acid sequence of SEQ ID NO: 442. In some embodiments, the TCR beta variable domain comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 453. In some embodiments, TCR beta variable domain comprises an amino acid sequence of SEQ ID NO: 453. In some embodiments, the TCR beta chain construct comprises a CDR1 having an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 428. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO: 428. In some embodiments, the TCR beta chain construct comprises a CDR1 having an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 443. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO: 443. In some embodiments, the TCR beta chain construct comprises a CDR2 having an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 429. In some embodiments, CDR2 comprises an amino acid sequence of SEQ ID NO: 429. In some embodiments, the TCR beta chain construct comprises a CDR2 having an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 444. In some embodiments, CDR2 comprises an amino acid sequence of SEQ ID NO: 444.

[0499] In some embodiments, the soluble TCR comprises the TCR alpha variable domain comprising a CDR1, a CDR2, and a CDR3. In some embodiments, the CDR1 has an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 422. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO: 422. In some embodiments, the CDR1 has an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 438. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO: 438. In some embodiments, the CDR1 has an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 448. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO:448. In some embodiments, the CDR2 comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 423. In some embodiments, the CDR2 comprises a sequence of SEQ ID NO: 423. In some embodiments, the CDR2 comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 239. In some embodiments, the CDR2 comprises a sequence of SEQ ID NO: 239. In some embodiments, the CDR2 comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 449. In some embodiments, the CDR2 comprises a sequence of SEQ ID NO: 449. In some embodiments, the CDR3 comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 424. In some embodiments, the CDR3 comprises a sequence of SEQ ID NO: 424. In some embodiments, the CDR3 comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 440. In some embodiments, the CDR3 comprises a sequence of SEQ ID NO: 440. In some embodiments, the CDR3 comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 450. In some embodiments, the CDR3 comprises a sequence of SEQ ID NO: 450. In some embodiments, the TCR alpha variable domain comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 421. In some embodiments, TCR alpha variable domain comprises an amino acid sequence of SEQ ID NO: 421. In some embodiments, the TCR alpha variable domain comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 437. In some embodiments, TCR alpha variable domain comprises an amino acid sequence of SEQ ID NO: 437. In some embodiments, the TCR alpha variable domain comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 447. In some embodiments, TCR alpha variable domain comprises an amino acid sequence of SEQ ID NO: 447.

[0500] In some embodiments, the soluble TCR comprises (a) the TCR beta variable domain having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to sequence of SEQ ID NO: 427, the TCR beta variable domain having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to sequence of SEQ ID NO: 434, the TCR beta variable domain having at least 60%, at least70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to sequence of SEQ ID NO: 442, or the TCR beta variable domain having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to sequence of SEQ ID NO: 456 and (b) the TCR alpha variable domain having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to sequence of SEQ ID NO: 421, the TCR alpha variable domain having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to sequence of SEQ ID NO: 437, or the TCR alpha variable domain having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to sequence of SEQ ID NO: 447.

[0501] In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 199. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 200. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 201. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 202. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 203. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 204. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 205. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 359. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 432. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the epitope is in complex with an MHC molecule encoded by an HLA allele. In some embodiments, the HLA allele is HLA-A*02:01. In some embodiments, the HLA allele is HLA-C*08:02.

[0502] In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 121, 127, 130, 534, 134, 136, 138, and 533. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 121. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 127. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 130. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 534. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 134. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 136. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 138. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 533.

[0503] In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 141-148 and 459-475. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 141. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 142. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 143. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%,70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 144. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 145. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 146. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 147. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 148. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 459. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 460. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 461. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 462. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 463. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 464. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 465. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 466. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 467. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 468. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 469. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 470. In someembodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 471. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 472. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 473. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 474. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 475.

[0504] In some embodiments, the soluble TCR comprises components summarized in Table 11. In some embodiments, the soluble TCR comprises a beta variable region with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 160, 178, 194, 236, 244, 252, 260, 268, 276, 284, 292, 300, 312, 327, 342, 356, 371, 384, 401, or 416. In some embodiments, the soluble TCR comprises a beta CDR1 with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 152, 170, 186, 232, 240, 248, 256, 264, 272, 280, 288, 296, 304, 319, 334, 348, 363, 378, 393, or 408. In some embodiments, the soluble TCR comprises a beta CDR2 with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 153, 171, 187, 233, 241, 249,257, 265, 273, 281, 289, 297, 305, 320, 335, 349, 364, 379, 394, or 409. In some embodiments, the soluble TCR comprises a beta CDR3 with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 154, 172, 188, 234, 242, 250,258, 266, 274, 282, 290, 298, 306, 321, 336, 350, 365, 380, 395, or 410. In some embodiments, the soluble TCR comprises an alpha variable region with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 157, 175, 191, 235, 243, 251, 259, 267, 275, 283, 291, 299, 309, 324, 339, 353, 368, 383, 398, or 413. In some embodiments, the TCR comprises an alpha CDR1 with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 149, 167, 183, 229, 237, 245, 253, 261, 269, 277, 285, 293, 301, 316, 331, 346, 360, 375, 390, or 405. In some embodiments, the TCR comprises an alpha CDR2 with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 150, 168, 184, 230, 238, 246, 254, 262, 270, 278, 286, 294, 302, 317, 332, 347, 361, 376, 391, or 406. In some embodiments, the TCR comprises an alpha CDR3 with at least 60%, 70%, 80%, 90%, 95%, or 100% sequenceidentity to any one of SEQ ID NOs: 151, 169, 185, 231, 239, 247, 255, 263, 271, 279, 287, 295, 303, 318, 333, 348, 362, 377, 392, or 407. In some embodiments, the soluble TCR recognizes an epitope with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NOs: 199 or 359.

[0505] The soluble TCR described herein can comprise a TCR alpha variable domain having a sequence having at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence selected from the group consisting of the TCR alpha variable domain sequences in Table 11, and / or a TCR beta variable domain having a sequence having at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence selected from the group consisting of the TCR beta variable domain sequences in Table 11. The soluble TCR described herein can comprise a TCR alpha chain having a sequence having at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence selected from the group consisting of the TCR alpha chain sequences in Table 11, and / or a TCR beta chain having a sequence having at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence selected from the group consisting of the TCR beta chain sequences in Table 11.

[0506] The soluble TCR described herein can comprise a TCR alpha variable domain having a sequence having at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence selected from the group consisting of the TCR alpha variable domain sequences (without the signal peptides) in Table 11, and / or a TCR beta variable domain having a sequence having at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence selected from the group consisting of the TCR beta variable domain sequences (without the signal peptides) in Table 11. The soluble TCR described herein can comprise a TCR alpha chain having a sequence having at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence selected from the group consisting of the TCR alpha chain sequences (without the signal peptides) in Table 11, and / or a TCR beta chain having a sequence having at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence selected from the group consisting of the TCR beta chain sequences (without the signal peptides) in Table 11.Table 11. KRAS TCRs Sequences (the signal peptides are bolded)

[0507] In some embodiments, the soluble TCR comprises components summarized in Table 12. In some embodiments, the soluble TCR comprises a beta variable region with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 12, 27, 42, 57, 72, 87, 102, 108, 110, 112, 114, 500, 502, or 504. In some embodiments, the soluble TCR comprises a beta CDR1 with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 4, 19, 34, 49, 64, 79, or 94. In some embodiments, the soluble TCR comprises a beta CDR2 with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 5, 20, 35, 50, 65, 80, or 95. In some embodiments, the soluble TCR comprises a beta CDR3 with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 6, 21, 36, 51, 66, 81, or 96. In some embodiments, the soluble TCR comprises an alpha variable region with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 9, 24, 39, 54, 69, 84, 99, 107, 109, 111, 113, 115, 501, or 503. In some embodiments, the TCR comprises an alpha CDR1 with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 1, 16, 31, 46, 61, 76, or 91. In some embodiments, the TCR comprises an alpha CDR2 with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 2, 17, 32, 47, 62, 77, or 92. In some embodiments, the TCR comprises an alpha CDR3 with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 3, 18, 33, 48, 63, 78, or 93. In some embodiments, the soluble TCR recognizes an epitope derived from a polypeptide having at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 485-487. These are summarized in Table 13.

[0508] The soluble TCR can comprise a TCR alpha variable domain having a sequence selected from the group consisting of the TCR alpha variable domain sequences in Table 12,and / or a TCR beta variable domain having a sequence selected from the group consisting of the TCR beta variable domain sequences in Table 12. The soluble TCR described herein can comprise a TCR alpha chain having a sequence selected from the group consisting of the TCR alpha chain sequences in Table 12, and / or a TCR beta chain having a sequence selected from the group consisting of the TCR beta chain sequences in Table 12.Table 12. GATA3 TCRs-Ill-Table 13 GATA3 neoORF SequencesCells

[0509] Provided herein is a cell comprising a chimeric antigen receptor (CAR). In some embodiments, the cell comprises a polynucleotide encoding the CAR. In some embodiments, the CAR comprises an extracellular domain comprising a domain that binds to an ALFA tag. In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the CAR comprises an intracellular domain. In some embodiments, the anti-tag binding domain comprises a CDR1 region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 477. In some embodiments, the CDR1 region has a sequence of SEQ ID NO: 477. In some embodiments, the anti-tag binding domain comprises a CDR2 region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 478. In some embodiments, the CDR2 region has a sequence of SEQ ID NO: 478. In some embodiments, the anti-tag binding domain comprises a CDR3 region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 479. In some embodiments, the CDR3 region has a sequence of SEQ ID NO: 479.

[0510] In some embodiments, the transmembrane domain is derived from CD28 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD3s, CD3^, or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD45 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD4 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD8 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD9 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD 16 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD22 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD33 or a functional variant thereof. In some embodiments, thetransmembrane domain is derived from CD37 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD64 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD80 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD86 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD 134 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD 154 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from KIRDS2 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from 0X40 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD2 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD27 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from LFA-1 (CD1 la or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD 18) or a functional variant thereof. In some embodiments, the transmembrane domain is derived from ICOS (CD278) or a functional variant thereof. In some embodiments, the transmembrane domain is derived from 4-1BB (CD137) or a functional variant thereof. In some embodiments, the transmembrane domain is derived from GITR or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD40 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from BAFFR or a functional variant thereof. In some embodiments, the transmembrane domain is derived from HVEM (LIGHTR) or a functional variant thereof. In some embodiments, the transmembrane domain is derived from SLAMF7 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from NKp80 (KLRF1) or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD 160 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD 19 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from IL2R beta or a functional variant thereof. In some embodiments, the transmembrane domain is derived from IL2R gamma or a functional variant thereof. In some embodiments, the transmembrane domain is derived from IL17Ra or a functional variant thereof. In some embodiments, the transmembrane domain is derived from ITGA1 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from VLA1 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD49a or afunctional variant thereof. In some embodiments, the transmembrane domain is derived from ITGA4 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from IA4 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD49D or a functional variant thereof. In some embodiments, the transmembrane domain is derived from ITGA6 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from VLA-6 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD49f or a functional variant thereof. In some embodiments, the transmembrane domain is derived from ITGAD or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD1 Id or a functional variant thereof. In some embodiments, the transmembrane domain is derived from ITGAE or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD 103 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from ITGAL or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD1 la or a functional variant thereof. In some embodiments, the transmembrane domain is derived from LFA-1 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from ITGAM or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD1 lb or a functional variant thereof. In some embodiments, the transmembrane domain is derived from ITGAX or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD11c or a functional variant thereof. In some embodiments, the transmembrane domain is derived from ITGB1 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD29 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from ITGB2 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD 18 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from LFA-1 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from ITGB7 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from TNFR2 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from DNAMI (CD226) or a functional variant thereof. In some embodiments, the transmembrane domain is derived from SLAMF4 (CD244 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from 2B4) or a functional variant thereof. In some embodiments, the transmembranedomain is derived from CD84 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD96 (Tactile) or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CEACAM1 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CRT AM or a functional variant thereof. In some embodiments, the transmembrane domain is derived from Ly9 (CD229) or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD160 (BY55) or a functional variant thereof. In some embodiments, the transmembrane domain is derived from PSGL1 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CDIOO (SEMA4D) or a functional variant thereof. In some embodiments, the transmembrane domain is derived from SLAMF6 (NTB-A or a functional variant thereof. In some embodiments, the transmembrane domain is derived from Lyl08) or a functional variant thereof. In some embodiments, the transmembrane domain is derived from SLAM (SLAMF1 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from CD 150 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from IPO-3) or a functional variant thereof. In some embodiments, the transmembrane domain is derived from BLAME (SLAMF8) or a functional variant thereof. In some embodiments, the transmembrane domain is derived from SELPLG (CD 162) or a functional variant thereof. In some embodiments, the transmembrane domain is derived from LTBR or a functional variant thereof. In some embodiments, the transmembrane domain is derived from PAG / Cbp or a functional variant thereof. In some embodiments, the transmembrane domain is derived from NKp44 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from NKp30 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from NKp46 or a functional variant thereof. In some embodiments, the transmembrane domain is derived from NKG2D or a functional variant thereof. In some embodiments, the transmembrane domain is derived from and NKG2C or a functional variant thereof.

[0511] In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain. In some embodiments, the extracellular domain is operably linked to the transmembrane domain by a hinge domain. In some embodiments, the hinge domain is a CD8a hinge domain. In some embodiments, the intracellular domain comprises an a primary cytoplasmic signaling sequence derived from CD3 combined with a costimulatory signaling region. In some embodiments or a functional variant thereof. In some embodiments, thecostimulatory signaling region is derived from the costimulatory signaling region is derived from CD28 or a functional variant thereof. In some embodiments, the costimulatory signaling region is derived from 0X40 or a functional variant thereof. In some embodiments, the costimulatory signaling region is derived from 4-1BB or a functional variant thereof. In some embodiments, the costimulatory signaling region is derived from ICOS or a functional variant thereof.

[0512] In some embodiments, the extracellular domain comprises an antibody or a functional fragment thereof. In some embodiments, the extracellular domain comprises a functional antibody fragment or a functional fragment thereof. In some embodiments, the extracellular domain comprises a single chain variable fragment (scFv) or a functional fragment thereof. In some embodiments, the extracellular domain comprises an Fab or a functional fragment thereof. In some embodiments, the extracellular domain comprises a single-domain antibody (sdAb) or a functional fragment thereof. In some embodiments, the extracellular domain comprises a nanobody or a functional fragment thereof. In some embodiments, the extracellular domain comprises a VH domain or a functional fragment thereof. In some embodiments, the extracellular domain comprises a VL domain or a functional fragment thereof. In some embodiments, the extracellular domain comprises a VNAR domain or a functional fragment thereof. In some embodiments, the extracellular domain comprises a VHH domain or a functional fragment thereof. In some embodiments, the extracellular domain comprises a bispecific antibody or a functional fragment thereof. In some embodiments, the extracellular domain comprises a diabody or a functional fragment thereof. In some embodiments, the extracellular domain comprises or a combination of the above.

[0513] In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain. In some embodiments, the CD8a transmembrane domain comprises a sequence with at least 80%, at least 85%, at least 90%, at least 95% identity to a sequence of SEQ ID NO: 480. In some embodiments, the CD8a transmembrane domain has a sequence of SEQ ID NO: 480. In some embodiments, the extracellular domain is operably linked to the transmembrane domain by a hinge domain. In some embodiments, the hinge domain is a CD8a hinge domain comprising a sequence with at least 80%, at least 85%, at least 90%, at least 95% identity to a sequence of SEQ ID NO: 490. In some embodiments, the CD8a hinge domain has a sequence of SEQ ID NO: 490. In some embodiments, the intracellular domain comprises a primary cytoplasmic signaling sequence derived from CD3 comprising a sequence with at least 80%, at least 85%, at least 90%, at least 95% identity to a sequence of SEQ ID NO: 482. In someembodiments, the primary cytoplasmic signaling sequence has a sequence of SEQ ID NO: 482. In some embodiments, the CAR further comprises a costimulatory signaling region derived from 4-1BB comprising a sequence with at least 80%, at least 85%, at least 90%, at least 95% identity to a sequence of SEQ ID NO: 481. In some embodiments, the costimulatory signaling sequence has a sequence of SEQ ID NO: 481. In some embodiments, the CAR comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 476. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 476. In some embodiments, the polynucleotide encoding the CAR comprises a sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 484. In some embodiments, the polynucleotide encoding the CAR comprises a sequence of SEQ ID NO: 484.

[0514] In some embodiments, the cell is an immune cell. In some embodiments, the cell is a myeloid cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a CD4 T cell. In some embodiments, the cell is a CD8 T cell.Pharmaceutical Compositions

[0515] Also provided herein are pharmaceutical compositions comprising the compositions and cells described above and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises any one of the tag-sTCRs described in the sTCR section and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises the polynucleotide encoding the tag-sTCR and a pharmaceutically acceptable carrier.

[0516] In some embodiments, the pharmaceutical composition comprises any one of the immune cell embodiments described in the previous sections, e.g., comprising the CAR and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises the immune cell comprising the polynucleotide encoding the CAR and a pharmaceutically acceptable carrier.

[0517] Pharmaceutical compositions can include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials can be non-toxic and may not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration.

[0518] Acceptable carriers, excipients, or stabilizers are those that are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3 -pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn- protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

[0519] Acceptable carriers are physiologically acceptable to the administered patient and retain the therapeutic properties of the compounds with / in which it is administered. Acceptable carriers and their formulations are generally described in, for example, Remington’ pharmaceutical Sciences (18th ed. A. Gennaro, Mack Publishing Co., Easton, PA 1990). One example of carrier is physiological saline. A pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject compounds from the administration site of one organ, or portion of the body, to another organ, or portion of the body, or in an in vitro assay system. Acceptable carriers are compatible with the other ingredients of the formulation and not injurious to a subject to whom it is administered. Nor should an acceptable carrier alter the specific activity of the neoantigens.

[0520] In one aspect, provided herein are pharmaceutically acceptable or physiologically acceptable compositions including solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration. Pharmaceutical compositions or pharmaceutical formulations therefore refer to a composition suitable for pharmaceutical use in a subject. Compositions can be formulated to be compatible with a particular route of administration(i.e., systemic or local). Thus, compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0521] In some embodiments, a composition can further comprise an acceptable additive in order to improve the stability of immune cells in the composition. Acceptable additives may not alter the specific activity of the immune cells. Examples of acceptable additives include, but are not limited to, a sugar such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose and mixtures thereof. Acceptable additives can be combined with acceptable carriers and / or excipients such as dextrose. Alternatively, examples of acceptable additives include, but are not limited to, a surfactant such as polysorbate 20 or polysorbate 80 to increase stability of the peptide and decrease gelling of the solution. The surfactant can be added to the composition in an amount of 0.01% to 5% of the solution. Addition of such acceptable additives increases the stability and halflife of the composition in storage.

[0522] The pharmaceutical composition can be administered, for example, by injection. Compositions for injection include aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal. Isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride can be included in the composition. The resulting solutions can be packaged for use as is, or lyophilized; the lyophilized preparation can later be combined with a sterile solution prior to administration. For intravenous, injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer’s Injection, Lactated Ringer’s Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included, as needed. Sterile injectable solutions can be prepared byincorporating an active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the active ingredient into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation can be vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0523] Compositions can be conventionally administered intravenously, such as by injection of a unit dose, for example. For injection, an active ingredient can be in the form of a parenterally acceptable aqueous solution which is substantially pyrogen-free and has suitable pH, isotonicity and stability. One can prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer’s Injection, Lactated Ringer’s Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included. Additionally, compositions can be administered via aerosolization.

[0524] When the compositions are considered for use in medicaments or any of the methods provided herein, it is contemplated that the composition can be substantially free of pyrogens such that the composition will not cause an inflammatory reaction or an unsafe allergic reaction when administered to a human patient. Testing compositions for pyrogens and preparing compositions substantially free of pyrogens are well understood to one or ordinary skill of the art and can be accomplished using commercially available kits.

[0525] Acceptable carriers can contain a compound that acts as a stabilizing agent, increases or delays absorption, or increases or delays clearance. Such compounds include, for example, carbohydrates, such as glucose, sucrose, or dextrans; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of peptides; or excipients or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents can also be used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers. To protect from digestion the compound can be complexed with a composition to render it resistant to acidic and enzymatic hydrolysis, or the compound can be complexed in an appropriately resistant carrier such as a liposome. Means of protecting compounds from digestion are known in the art (e.g., Fix (1996) Pharm Res. 13: 1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48: 119 135; and U.S. Pat. No. 5,391,377).

[0526] The compositions can be administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered can depend on the subject to be treated, capacity of the subject’s immune system to utilize the active ingredient, and degree of binding capacity desired. Precise amounts of active ingredient required to be administered can depend on the judgment of the practitioner and can be peculiar to each individual. Suitable regimes for initial administration and booster shots can also be variable but can be typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusions sufficient to maintain concentrations in the blood can be contemplated.

[0527] A pharmaceutical composition can be encapsulated within liposomes using well- known technology. Biodegradable microspheres can also be employed as carriers for the pharmaceutical compositions of this present disclosure.

[0528] The pharmaceutical composition can be administered in liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to a patient are well known to those of skill in the art. Essentially, material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary.

[0529] Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the blood stream. Alternatively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time ranging from days to months.

[0530] A pharmaceutical composition comprising an active agent such as an immune cell described herein, in combination with one or more adjuvants can be formulated in conventional manner using one or more physiologically acceptable carriers, comprising excipients, diluents, and / or auxiliaries, e.g., which facilitate processing of the active agents into preparations that can be administered. Proper formulation can depend at least in part upon the route of administration chosen. The agent(s) described herein can be delivered to a patient using a number of routes or modes of administration, including oral, buccal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous, and intramuscular applications, as well as by inhalation.

[0531] The active agents can be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and can be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi -dose containers with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol.

[0532] In some embodiments, the pharmaceutical composition comprises a preservative or stabilizer. In some embodiments the preservative or stabilizer is selected from a cytokine, a growth factor or an adjuvant or a chemical substance. In some embodiments, the composition comprises at least one agent that helps preserve cell viability through at least one cycle of freeze-thaw. In some embodiments, the composition comprises at least one agent that helps preserve cell viability through at least more than one cycle of freeze-thaw.

[0533] For injectable formulations, the vehicle can be chosen from those known in art to be suitable, including aqueous solutions or oil suspensions, or emulsions, with sesame oil, com oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. The formulation can also comprise polymer compositions which are biocompatible, biodegradable, such as poly(lactic-co-glycolic)acid. These materials can be made into micro or nanospheres, loaded with drug and further coated or derivatized to provide superior sustained release performance. Vehicles suitable for periocular or intraocular injection include, for example, suspensions of therapeutic agent in injection grade water, liposomes and vehicles suitable for lipophilic substances. Other vehicles for periocular or intraocular injection are well known in the art.

[0534] In some instances, pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0535] Pharmaceutical compositions comprising, for example, an active agent such as immune cells disclosed herein, in combination with one or more adjuvants can be formulated to comprise certain molar ratios. For example, molar ratios of about 99: 1 to about 1 :99 of an active agent such as an immune cell described herein, in combination with one or more adjuvants can be used. In some instances, the range of molar ratios of an active agent such as an immune cell described herein, in combination with one or more adjuvants can be selected from about 80:20 to about 20:80; about 75:25 to about 25:75, about 70:30 to about 30:70, about 66:33 to about 33:66, about 60:40 to about 40:60; about 50:50; and about 90: 10 to about 10:90. The molar ratio of an active agent such as an immune cell described herein, in combination with one or more adjuvants can be about 1 :9, and in some cases can be about 1 : 1. The active agent such as an immune cell described herein, in combination with one or more adjuvants can be formulated together, in the same dosage unit e.g., in one vial, suppository, tablet, capsule, an aerosol spray; or each agent, form, and / or compound can be formulated in separate units, e.g., two vials, suppositories, tablets, two capsules, a tablet and a vial, an aerosol spray, and the like.

[0536] In some embodiments, the pharmaceutical composition comprises a nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle or a liposome. In some embodiments, the particle is a lipoplex. In some embodiments, the lipid is comprised in a vesicle encapsulating said RNA. The vesicle may be a multilamellar vesicle, an unilamellar vesicle, or a mixture thereof. The vesicle may be a liposome.

[0537] In some embodiments, the nanoparticles are lipoplexes comprising DOTMA and DOPE in a molar ratio of 10:0 to 1 :9, in some cases 8:2 to 3:7, and in some cases of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, in some cases 1.6:2 to 1 :2, in some cases 1.4:2 to 1.1 :2 and in some cases about 1.2:2.

[0538] In some embodiments, the nanoparticles are lipoplexes comprising DOTMA and Cholesterol in a molar ratio of 10:0 to 1 :9, in some cases 8:2 to 3:7, and in some cases of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, in some cases 1.6:2 to 1 :2, even in some cases 1.4:2 to 1.1 :2 and in some cases about 1.2:2.

[0539] In some embodiments, the nanoparticles are lipoplexes comprising DOTAP and DOPE in a molar ratio of 10:0 to 1 :9, in some cases 8:2 to 3:7, and in some cases of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNAis 1.8:2 to 0.8:2, in some cases 1.6:2 to 1 :2, in some cases 1.4:2 to 1.1 :2 and in some cases about 1.2:2.

[0540] In some embodiments, the nanoparticles are lipoplexes comprising DOTMA and DOPE in a molar ratio of 2: 1 to 1 :2, in some cases 2: 1 to 1 : 1, and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.4: 1 or less.

[0541] In some embodiments, the nanoparticles are lipoplexes comprising DOTMA and cholesterol in a molar ratio of 2: 1 to 1 :2, in some cases 2: 1 to 1 : 1, and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.4: 1 or less. In some embodiments, the nanoparticles are lipoplexes comprising DOTAP and DOPE in a molar ratio of 2: 1 to 1 :2, in some cases 2: 1 to 1 : 1, and wherein the charge ratio of positive charges in DOTAP to negative charges in the RNA is 1.4: 1 or less.

[0542] In some embodiments, the nanoparticles have an average diameter in the range of from about 50 nm to about 1000 nm, in some cases from about 50 nm to about 400 nm, in some cases about 100 nm to about 300 nm such as about 150 nm to about 200 nm. In some embodiments, the nanoparticles have a diameter in the range of about 200 to about 400 nm.

[0543] In some embodiments, the poly dispersity index of the nanoparticles described herein as measured by dynamic light scattering is 0.5 or less, in some cases 0.4 or less or in some cases 0.3 or less.

[0544] In some embodiments, the nanoparticles described herein are obtainable by one or more of the following: (i) incubation of liposomes in an aqueous phase with the RNA in an aqueous phase, (ii) incubation of the lipid dissolved in an organic, water miscible solvent, such as ethanol, with the RNA in aqueous solution, (iii) reverse phase evaporation technique, (iv) freezing and thawing of the product, (v) dehydration and rehydration of the product, (vi) lyophilization and rehydration of the of the product, or (vii) spray drying and rehydration of the product.

[0545] In some embodiments, the nanoparticles are produced by a process comprising a step of incubating the RNA with bivalent cations in some cases at a concentration of between 0.1 mM to 5 mM such as 0.1 mM to 4 mM or 0.3 mM to 1 mM prior to incorporation into said nanoparticles and / or by incubating the RNA with monovalent ions in some cases at a concentration of between 1 mM to 500 mM such as 100 mM to 200 mM or 130 mM to 170 mM prior to incorporation into said nanoparticles and / or by incubating the RNA with buffers prior to incorporation into said nanoparticles.

[0546] In some embodiments, after incubation of the bivalent cations to RNA a step of dilution by adding liposomes and / or other aqueous phases by at least a factor of more than 1.5, in some cases by a factor of more than 2, or by a factor of more than 5 is involved. In some embodiments, the bivalent cations are calcium ions, where the final concentration of said calcium ions is less than 4 mM, in some cases less than 3 mM and in some cases 2.2 mM or less.

[0547] In some embodiments, the nanoparticles described herein are produced by a process comprising a step of extruding and / or a step of filtration and / or a step of lyophilizing the nanoparticles.Use and Methods of Treatment

[0548] The compositions or cells provided herein can be used in the manufacture of a medicament in treating a disease or condition in a subject in need thereof.

[0549] The methods of the disclosure can be used to treat any type of cancer known in the art. Non-limiting examples of cancers to be treated by the methods of the present disclosure can include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), cutaneous melanoma, synovial sarcoma, myxoid and round cell liposarcoma, osteosarcoma, and neuroblastoma , esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies.

[0550] Additionally, the disease or condition provided herein includes refractory or recurrent malignancies whose growth may be inhibited using the methods of treatment of the present disclosure. In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is selected from the group consisting of carcinoma, squamous carcinoma, adenocarcinoma, sarcomata, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, squamous cell carcinoma of the anogenital region, melanoma and renal cell carcinoma.

[0551] Specific examples of cancers that can be prevented and / or treated in accordance with present disclosure include, but are not limited to, the following: renal cancer, kidney cancer, glioblastoma multiforme, metastatic breast cancer; breast carcinoma; breast sarcoma; neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma; malignant melanoma; carcinomas of the epidermis; leukemias such as but not limited to, acute leukemia, acutelymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin’s disease, non-Hodgkin’s disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma;Waldenstrom’s macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone cancer and connective tissue sarcomas such as but not limited to bone sarcoma, myeloma bone disease, multiple myeloma, cholesteatoma-induced bone osteosarcoma, Paget’s disease of bone, osteosarcoma, chondrosarcoma, Ewing’s sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi’s sarcoma, leiomyosarcoma, liposarcoma, lymphangio sarcoma, neurilemmoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget’s disease (including juvenile Paget’s disease) and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as but limited to Cushing’s disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and cilliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget’s disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to,ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; cervical carcinoma; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; colorectal cancer, colon carcinoma; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to pappillary, nodular, and diffuse; lung cancers such as non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; lung carcinoma; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, androgen-independent prostate cancer, androgen-dependent prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acrallentiginous melanoma; kidney cancers such as but not limited to renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and / or uterus); renal carcinoma; Wilms’ tumor; bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas. Cancers include, but are not limited to, B cell cancer, e.g., multiple myeloma, Waldenstrom’s macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lungcancer, bronchus cancer, colorectal cancer, prostate cancer (e.g., metastatic, hormone refractory prostate cancer), pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, and the like. Other nonlimiting examples of types of cancers applicable to the methods encompassed by the present disclosure include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin’s disease and non-Hodgkin’s disease), multiple myeloma, Waldenstrom’s macroglobulinemia, and heavy chain disease. In some embodiments, the cancer whose phenotype is determined by the method of the present disclosure is an epithelial cancer such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. Theepithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, brenner, or undifferentiated. In some embodiments, the present disclosure is used in the treatment, diagnosis, and / or prognosis of lymphoma or its subtypes, including, but not limited to, mantle cell lymphoma. Lymphoproliferative disorders are also considered to be proliferative diseases.

[0552] Cancer refers to diseases in which abnormal cells divide out of control and are able to invade other tissues. Cancer cells can spread to other parts of the body through the blood and lymph systems. Cancer can be characterized as a group of diseases involving abnormal cell growth that may begin in any tissue with the potential to invade or spread to other parts of the body. Some cancers can be characterized by their type, e.g., solid cancers, liquid cancers, or based on cellular origin such as hematopoietic cancers, osteosarcoma or lymphoma. Some cancers are known by the tissue of their origin or prevalence, e.g., endometrial cancers are characterized as cancers of the endometrial tissue. Some cancers are known by the organ or site of their origin or prevalence, e.g. lung cancer, head and neck cancer. Some cancers may be known by the overproductions of certain proteins, enzymes or biomarkers compared to their counterpart cells or tissues that are not cancerous. For example, certain proteins of viral origin may be associated with certain cancers, such as HPV-16 cancers, where certain proteins, for example HPV-16 E6 and E7 are overexpressed in cancer cells of this type. For example, certain antigens, such as KRAS may be highly expressed in certain cancer types, compared to non-cancer cells of the same type, and may be designated as KRAS overexpressing cancers. Typically, the overexpression of the antigen or the specific protein may be associated with or related to one or more mutations, and the cancer type may be associated with the mutation. For example, mutation at the wild type G residue corresponding to position 12 in KRAS amino acid sequence may be mutated to V, D, C or other amino acids in KRAS-specific cancer cells. Certain specific antigens may be specifically expressed in cancer cells of certain cancer types, and not in other cancer types. Various cancer are contemplated herein that may not be restricted to a specific cell type, tissue type or organ, or even a certain stage of cancer. The TCRs of the present invention are directed to cancer cells that express a cancer antigen, that may be patient specific, which can be found during sequencing of a subject’s genome from biological sample obtained from a cancer cell, cancer site or cancer tissue and compared to a corresponding non-cancer sample from the same subject; wherein the patient-specific antigen may be expressed in the cancer cell, and not on the non-cancer cell of the subject. In some cases, cancer antigens may be cancer specific,where the antigen is reportedly present in the type of cancer observed in multiple patients in the human population, who have been diagnosed of the specific cancer. In some cases, certain types are cancers are associated with an antigen, a protein (e.g., a viral protein) a gene mutation, all forms of cancer are contemplated herein.

[0553] In some embodiments, the cancer is a solid cancer. In some cases, the cancer is a liquid / blood cancer. The cancer can express or be diagnosed as expressing a tumor antigen. The tumor antigen can be a tumor-associated antigen or a tumor-specific antigen. In some cases, the cancer expresses a tumor-associated antigen (TAA). In some cases, the cancer expresses a tumor-specific antigen (TSA).

[0554] In some embodiments, the cancer is a cancer expressing or diagnosed as expressing a TAA. In some embodiments, the cancer is a cancer expressing or diagnosed as expressing a TSA.

[0555] The current classification of TAA can include the following group: a) Cancer testis (CT) antigen: Since testis cells do not express HLA class I and class II molecules, these antigens may not be recognized by T cells in normal tissues and may therefore be immunologically considered tumor specific. Non-limiting examples of CT antigens include members of the MAGE family and NY-ESO-1; b) Differentiation antigen: both tumor and normal tissue (from which the tumor originates) may contain TAAs. Differentiation antigens may be found, for example, in melanoma and normal melanocytes. Many of these melanocyte lineage-associated proteins may be involved in melanin biosynthesis and therefore these proteins may not tumor-specific but may still widely be used for immunotherapy of cancer. Examples include, but are not limited to, tyrosinase for melanoma and PSA for Melan-A / MART-1 or prostate cancer; c) Overexpressed TAA: gene-encoded widely expressed TAAs may be detected in histologically diverse tumors and in many normal tissues, with generally low expression levels. It is possible that many epitopes processed and potentially presented by normal tissues may be below the threshold level of T cell recognition, whereas their overexpression in tumor cells can trigger anti cancer responses by breaking previously established tolerance. Non-limiting examples of such TAAs include Her-2 / neu, survivin, telomerase or WT1; d) tumor specific antigen can include unique TAAs resulted from mutations in normal genes (e.g., beta-catenin, CDK4). Some of these molecular changes can be associated with neoplastic transformation and / or progression. Tumor-specific antigens can generally induce strong immune responses without risking from the autoimmune response to normal tissuestrips. On the other hand, these TAAs may only be associated with the exact tumor on which they are confirmed and may not commonly shared among many individual tumors. In the case of tumor specific (related) isoform proteins, peptide tumor specificity (or relatedness) may also occur if the peptide is derived from tumor (related) exons; e) TAA resulting from aberrant post-translational modification: such TAAs may result from proteins in the tumor that are neither specific nor overexpressed, but which still have tumor relevance (this relevance is due to posttranslational processing that is primarily active on tumors). Such TAAs may result from an altered glycosylation pattern, resulting in a tumor producing a novel epitope for MUC1 or in an event such as protein splicing during degradation, which may or may not be tumor specific; and f) Tumor virus protein: these TTAs are viral proteins that may play a key role in the oncogenic process and, because they are foreign proteins (non-human proteins), may be able to trigger T cell responses. Non-limiting examples of such proteins include human papilloma type 16 viral proteins, E6 and E7, which are expressed in cervical cancer.

[0556] Examples of tumor antigens include, but not limited to new antigens expressed during tumorigenesis, products of oncogenes and tumor suppressor genes, overexpressed or abnormally expressed intracellular proteins (e.g., HER2, MUC1, PSA, MUC1), carcinoembryonic antigen (CEA), tumor viruses (e.g., EBC, HPV, HBV, HCB, HTLV), cancer testis antigens (CTA) (e.g., MAGE family, NY-ESO), oncofetal antigens, altered surface glycolipids and glycoproteins, cell type-specific differentiation antigens (e.g., MART-1), or a derivative thereof. The tumor antigens can be selected from the group consisting of NY-ESO-1, Her2 / neu, SSX-2, MAGE-C2, MAGE-A1, M-2433-233, MAGE- A10 254-262, KK-LC-1, p53, PRAME, Alpha fetoprotein, HPV6-E6, HPV16-E7, EBV- LMP1, RAS: G12D, RAS: G12C, RAS: G12A, RAS: G12S, RAS: G12R, RAS: G12R, RAS: G12R, RAS: G122 V, RAS: Q61H, RAS: Q61L, RAS: Q61R, RAS: G13D, TP53: V157G, TP53: V157F, TP53: R248Q, TP53: R248W, TP53: G245S, TP53: Y163C, TP53: G249S, TP53: Y240C, TP53: R175H, TP53: K132N, CDC73: Q254E, TPP2A6: N438Y, CTNN1 : T41A, CTNNB1 : S45P, CTNNB1 : S37Y, CTNNB1 : S33C, EGFR: L858R, EGFR: T790M, PIK3CA: E542K, PIK3CA: H1047R, GNAS: R201H, CDK4:R24, R24C H3. 3:K28M, BRAF: V600E, CHD4 K73Rfs, NRAS Q61R, IDH1 :R132H, TVP23C: C51Y, and any combination thereof. The RAS can be KRAS, HRAS, or NRAS.

[0557] Other non-limiting examples of tumor-associated antigen or tumor-specific antigen includes antigens from Human Papilloma Virus, Epstein-Barr Virus, Merkel cellpolyomavirus, Human Immunodeficiency Virus, Human T-cell Leukemia Virus, Human Herpes Virus 8, Hepatitis B virus, Hepatitis C virus, HCV, HBC, Cytomegalovirus, or from the group of single-point mutated antigens derived from the group consisting of the antigens of ctnnbl gene, casp8 gene, HER2 gene, p53 gene, KRAS gene, NRAS gene, or particular tumor antigens issued or derived from the group consisting of RAS oncogene, BCR-ABL tumor antigens, ETV6-AML1 tumor antigens, melanoma-antigen encoding genes (MAGE), BAGE antigens, GAGE antigens, ssx antigens, ny-eso-1 antigens, cyclin-Al tumor antigens, MART-1 antigen, gplOO antigen, CD 19 antigen, prostate specific antigen, prostatic acidic phosphatase antigen, carcinoembryonic antigen, alphafetoprotein antigen, carcinoma antigen 125, mucin 16 antigen, mucin 1 antigen, human telomerase reverse transcriptase antigen, EGFR antigen, MOK antigen, RAGE-1 antigen, PRAME antigen, wild-type p53 antigen, oncogene ERBB2 antigen, sialyl-Tn tumor antigen, Wilms tumor 1 antigen, mesothelin antigen, carbohydrate antigens, B-catenin antigen, MUM-1 antigen, CDK4 antigen ERBB2IP antigen, and Melan-A melanoma tumor-associated antigen.

[0558] In some cases, the cancer cells express the tumor antigens, including and not limited to, NY-ESO-1, Her2 / neu, SSX-2, MAGE-C2, MAGE-A1, M-2433-233, MAGE-A10 254- 262, KK-LC-1, p53, PRAME, Alpha fetoprotein, HPV6-E6, HPV16-E7, EBV-LMP1, RAS: G12D, RAS: G12C, RAS: G12A, RAS: G12S, RAS: G12R, RAS: G12R, RAS: G12R, RAS: G122 V, RAS: Q61H, RAS: Q61L, RAS: Q61R, RAS: G13D, TP53: V157G, TP53: V157F, TP53: R248Q, TP53: R248W, TP53: G245S, TP53: Y163C, TP53: G249S, TP53: Y240C, TP53: R175H, TP53: K132N, CDC73: Q254E, TPP2A6: N438Y, CTNN1 : T41A, CTNNB1 : S45P, CTNNB1 : S37Y, CTNNB1 : S33C, EGFR: L858R, EGFR: T790M, PIK3CA: E542K, PIK3CA: H1047R, GNAS: R201H, CDK4:R24, R24C H3. 3:K28M, BRAF: V600E, CHD4 K73Rfs, NRAS Q61R, IDH1 :R132H, or TVP23C: C51Y. The RAS can be KRAS, HRAS, or NRAS.

[0559] In some embodiments, the cancer is a carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, head and neck cancer, colorectal cancer,rectal cancer, soft-tissue sarcoma, Kaposi’s sarcoma, B-cell lymphoma (including low grade / follicular non-Hodgkin’s lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade / follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom’s macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myeloma, Hairy cell leukemia, chronic myeloblasts leukemia, and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema, Meigs’ syndrome, or combinations thereof.

[0560] Provided herein is a method of treating a disease or condition in a subject in need thereof. In some embodiments, the method comprises administering a composition comprising a soluble TCR. In some embodiments, the method comprises administering a therapeutically effective population of cells comprising a CAR or a polynucleotide encoding the CAR.

[0561] Also provided herein is a method of treating a disease or condition in a subject in need thereof. In some embodiments, the method comprises (a) administering a soluble TCR comprising a tag (tag-sTCR) or a polynucleotide encoding the tag-sTCR to a subject in need thereof, wherein the tag-sTCR binds to a target cell in the subject. In some embodiments, the method comprises (b) administering a therapeutically-effective population of immune cells comprising a chimeric receptor (CAR) or polynucleotide encoding the CAR to the subject. In some embodiments, administering in (a) is prior to administering in (b). In some embodiments, administering in (a) is after administering in (b). In some embodiments, administering in (a) is concurrent with administering in (b).

[0562] In some embodiments, the soluble TCR has at least one tag (tag-sTRCR). In some embodiments, the composition comprises a polynucleotide encoding the tag-sTCR. In some embodiments, the immune cell comprises a CAR. In some embodiments, the CAR comprises an extracellular domain comprising an anti-tag binding domain. In some embodiments, the anti-tag binding domain binds to the at least one tag of the tag-sTCR. In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the CAR comprises an intracellular domain. In some embodiments, the CAR is expressed on the surface of a cell. In some embodiments the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD4 T cell. In some embodiments, the T cell is aCD8 T cell. In some embodiments, the tag-sTCR binds to a peptide:MHC complex of a target cell. In some embodiments, the target cell is a cancer cell.

[0563] In some embodiments, the soluble TCR comprises a peptide:MHC complex engager. In some embodiments, the peptide:MHC complex engager comprises a TCR alpha variable domain. In some embodiments, the peptide:MHC complex engager comprises a TCR beta variable domain. In some embodiments, the peptide:MHC complex engager comprises a TCR alpha variable domain and a TCR beta variable domain.

[0564] In some embodiments, the soluble TCR is not a single chain TCR. In some embodiments, the soluble TCR does not comprise a constant region of a TCR.

[0565] In some embodiments, the soluble TCR is a single chain TCR. In some embodiments, the TCR alpha variable domain is operably linked to the TCR beta variable domain. In some embodiments, the soluble TCR comprises, form N-terminus to C-terminus, the TCR alpha variable domain operably linked to the TCR beta variable domain. In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the TCR beta variable domain operably linked to the TCR alpha variable domain.

[0566] In some embodiments, the tag-sTCR is a fusion protein. In some embodiments, the at least one tag is operably linked to the soluble TCR. In some embodiments, the at least one tag is operably linked to the N-terminus of the soluble TCR. In some embodiments, the at least one tag is operably linked to the C-terminus of the soluble TCR.

[0567] In some embodiments, the soluble TCR is a single chain TCR. In some embodiments, the TCR comprises, from N-terminus to C-terminus, the TCR beta variable domain, the TCR beta constant domain, the TCR alpha variable domain, and the TCR alpha constant domain. In some embodiments, the at least one tag is operably linked to the N-terminus of the soluble TCR. In some embodiments, the soluble TCR comprises an anti-CD3 ScFv region operably linked to the N-terminus of the soluble TCR. In some embodiments, the soluble TCR comprises a linker. In some embodiments, the linker is operably linked to the TCR alpha constant domain and the TCR alpha variable domain. In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10. In some embodiments, n is 7. In some embodiments, the soluble TCR is a single chain TCR. In some embodiments, the TCR comprises from N-terminus to C-terminus, the TCR alpha variable domain, the TCR alpha constant domain, the TCR alpha variable domain, and the TCR beta constant domain. In some embodiments, the at least one tag is operably linked to the TCR beta variable domain. In some embodiments, the soluble TCR comprises an anti-CD3 ScFvregion operably linked to the TCR beta variable domain. In some embodiments, the soluble TCR comprises a linker. In some embodiments, the linker is operably linked to the at least one tag or the anti-ScFv region and the TCR beta constant domain. In some embodiments, the linker is operably linked to the at least one tag and the TCR beta constant domain. In some embodiments, the linker is operably linked to the anti-ScFv region and the TCR beta constant domain. In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10. In some embodiments, n is 4. In some embodiments, the soluble TCR comprises an additional tag operably linked to the C-terminus of the soluble TCR. In some embodiments, the additional tag is a lOXHIS-sortag operably linked to the C-terminus of the soluble TCR. In some embodiments, the soluble TCR comprises a lOXHIS-sortag operably linked to the C-terminus of the soluble TCR. In some embodiments, the soluble TCR comprises one or more disulfide bridges between the TCR alpha constant domain and the TCR beta constant domain. In some embodiments, the soluble TCR comprises a modification comprising removal of one or more N-glycosylation sites in the TCR alpha constant domain and / or TCR beta constant domain. In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the TCR alpha variable domain, the TCR alpha constant domain, the linker, the at least one tag, the TCR beta variable region, the TCR beta constant region, and the lOXHIS-sortag. In some embodiments, the soluble TCR comprises, from N-terminus to C-terminus, the at least one tag, the TCR beta variable domain, the TCR beta constant domain, the linker, the TCR alpha variable domain, the TCR alpha constant domain, and the lOXHIS-sortag.

[0568] In some embodiments, the TCR alpha variable domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from the group consisting of the TCR alpha variable domain sequences of Tables 5, 6, 8, 11, and 12. In some embodiments, the TCR beta variable domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from the group consisting of the TCR beta variable domain sequences of Tables 5, 6, 8, 11, and 12. In some embodiments, the TCR alpha constant domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from the group consisting of the TCR alpha constant domain sequences of Tables 5, 6, 8, 11, and 12. In some embodiments, the TCR beta constant domain comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selectedfrom the group consisting of the TCR beta constant domain sequences of Tables 5, 6, 8, 11, and 12. In some embodiments, wherein the additional tag comprises a sequence having at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% identify to a sequence of SEQ ID NO: 131. In some embodiments, wherein the additional tag comprises a sequence of SEQ ID NO: 131.

[0569] In some embodiments, the at least one tag binds to the anti-tag binding domain of the CAR. In some embodiments, the anti-tag binding domain is an antibody or antigen binding fragment thereof.

[0570] In some embodiments, the at least one tag is not an affinity tag. In some embodiments, the at least one tag does not comprise a fluorophore. In some embodiments, the tag-sTCR does not comprise a scFv. In some embodiments, the at least one tag is a polypeptide that is absent in eukaryotic systems.

[0571] In some embodiments, the at least one tag comprises less than 500, less than 450, less than 400, less than 350 , less than 300, less than 250 , less than 200, less than 150, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, less than 10, or less than 5 amino acids. In some embodiments, the at least one tag comprises at least 1, at least 2, at least 3 , at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, or at least 300 or more amino acids.

[0572] In some embodiments, the at least one tag does not comprise modified amino acids. In some embodiments, the at least one tag does not comprise unnatural amino acids. In some embodiments, the at least one tag does not comprise modified amino acids or unnatural amino acids. In some embodiments, all amino acids of the at least one tag are non-modified amino acids with natural peptide bonds. In some embodiments, the at least one tag forms a stable alpha-helical structure. In some embodiments, the at least one tag is hydrophilic. In some embodiments the at least one tag is neutral at physiological pH. In some embodiments, the at least one tag is hydrophilic and is neutral at physiological pH. In some embodiments, the at least one tag comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 126. In some embodiments, the at least one tag comprises a sequence of SEQ ID NO: 126. In some embodiments, the at least one tag comprises a sequence that comprises less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 amino acidsdiffering from SEQ ID NO: 126. In some embodiments, the at least one tag further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more amino acids on the N-terminus of the sequence. In some embodiments, the at least one tag further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more amino acids on the C-terminus of the sequence. In some embodiments, the at least one tag further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more amino acids on both the N-terminus and C- terminus of the sequence.

[0573] In some embodiments, the at least one tag is recognized by the anti-tag binding domain of the CAR with a Ka of less than 3,000 nM, less than 2,500 nM, less than 2,000 nM, less than 1,500 nM, less than 1,000 nM, less than 500 nM, less than 250 nM, less than 150 nM, less than 100 nM, less than 90 nM, 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, or less than 1 nM. In some embodiments, the peptide:MHC complex engager specifically binds to an endogenous disease specific antigen. In some embodiments, the peptide:MHC complex engager specifically binds to a cancer antigen. In some embodiments, the cancer antigen is a neoantigen. In some embodiments, the cancer antigen is a tumor associated antigen. In some embodiments, the cancer antigen is a viral antigen. In some embodiments, the peptide:MHC complex engager binds to the peptide:MHC complex comprising an epitope from the endogenous disease specific antigen that is presented on the surface of the target cell. In some embodiments, the target cell is a cancer cell.

[0574] In some embodiments, the peptide:MHC complex engager binds to the peptide:MHC complex with a Ka of less than 3,000 nM, less than 2,500 nM, less than 2,000 nM, less than 1,500 nM, less than 1,000 nM, less than 500 nM, less than 250 nM, less than 150 nM, less than 100 nM, less than 90 nM, 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, or less than 1 nM. In some embodiments, the MHC of the peptide:MHC complex is a class I MHC. In some embodiments, the MHC of the peptide:MHC complex is a class II MHC.

[0575] In some embodiments, the epitope is derived from a RAS polypeptide or a fragment thereof. In some embodiments, the RAS polypeptide is a mutated RAS polypeptide. In some embodiments, the epitope derived from the RAS polypeptide or a fragment thereof comprises a G12C mutation. In some embodiments, the epitope derived from the RAS polypeptide or a fragment thereof comprises a G12V mutation. In some embodiments, the epitope derived from the RAS polypeptide or a fragment thereof comprises a G12D mutation. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with atleast 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 199. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 199. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 200. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 200. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 201. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 201. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 202. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 202. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 203. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 203. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 204. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 204. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 205. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 205. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 359. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 359. In some embodiments the peptide:MHC complex comprises an epitope having a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 432. In some embodiments, the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NO: 432. In some embodiments, an MHC of the peptide:MHC complex comprising the epitope derived from the RAS polypeptide or a fragment thereof comprises an MHC encoded by an HLA-A*02:01 allele. . In some embodiments, an MHC ofthe peptide:MHC complex comprising the epitope derived from the RAS polypeptide or a fragment thereof comprises an MHC encoded by an HLA-C*08:02 allele.

[0576] In some embodiments, the epitope is derived from a GATA3 polypeptide or a fragment thereof. In some embodiments, the GATA3 polypeptide is a mutated GATA3 polypeptide. In some embodiments, the GATA3 polypeptide is encoded by a neoORF. In some embodiments, the epitope derived from a GAT A3 polypeptide or a fragment thereof has a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the epitope has a sequence of SEQ ID NO: 15.

[0577] In some embodiments, the epitope is derived from a Human Papillomavirus (HPV) protein. In some embodiments, the HPV protein is a HPV16 E7 protein. In some embodiments, the epitope has a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 458. In some embodiments, the epitope has a sequence of SEQ ID NO: 458.

[0578] In some embodiments, the single chain TCR comprises a linker. In some embodiments, the linker is between the TCR alpha variable domain and the TCR beta variable domain. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker comprises the at least one tag sequence. In some embodiments, the linker comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 125. In some embodiments, the linker has a sequence of SEQ ID NO: 125. In some embodiments, the linker comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 128. In some embodiments, the linker has a sequence of SEQ ID NO: 128. In some embodiments, the linker comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 135. In some embodiments, the linker has a sequence of SEQ ID NO: 135. In some embodiments, the linker comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 488. In some embodiments, the linker has a sequence of SEQ ID NO: 488.

[0579] In some embodiments, the soluble TCR is a single chain TCR, and wherein the single chain TCR comprises, from N terminus to C terminus, the TCR alpha variable domain, the TCR alpha constant domain, a first linker, a first tag, a second linker, a second tag, the TCR beta variable domain, and the TCR beta constant domain. In some embodiments, the first tagor the second tag comprises a sequence of SEQ ID NO: 126. In some embodiments, the first tag and the second tag each comprises a sequence of SEQ ID NO: 126. In some embodiments, the first linker or the second linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10. In some embodiments, the integer is 1. In some embodiments, the integer is 2. In some embodiments, the integer is 3. In some embodiments, the integer is 4. In some embodiments, the integer is 5. In some embodiments, the integer is 6. In some embodiments, the integer is 7. In some embodiments, the integer is 8. In some embodiments, the integer is 9. In some embodiments, the integer is 10. In some embodiments, the first linker comprises a sequence of (GGGGS)4, and the second linker comprises a sequence of GGGGS. In some embodiments, the first linker comprises a sequence of (GGGGS)2, and the second linker comprises a sequence of GGGGS.

[0580] In some embodiments, the soluble TCR comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 552, 556, and 557. In some embodiments, the soluble TCR comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO: 552. In some embodiments, the soluble TCR comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO: 556. In some embodiments, the soluble TCR comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO: 557.

[0581] In some embodiments, the TCR alpha variable domain and the TCR beta variable domain are on two separate polypeptide chains comprising a first polypeptide chain and a second polypeptide chain. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 138, 541, 543, 544, 545, 547, 548, and 550. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 138. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 541. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 543. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 544. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 545. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 547. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 548. In some embodiments, the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence of SEQ ID NO: 550.

[0582] In some embodiments, the first polypeptide chain comprises a first dimerizing domain. In some embodiments, the first dimerizing domain is fused to the TCR alpha variable domain. In some embodiments, the second polypeptide chain comprises a second dimerizing domain. In some embodiments, the second dimerizing domain is fused to the TCR beta variable domain. In some embodiments, the first dimerizing domain comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25 or more amino acids of a TCR extracellular domain adjacent to a TCR transmembrane domain. In some embodiments, the second dimerizing domain comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25 or more amino acids of a TCR extracellular domain adjacent to a TCR transmembrane domain.

[0583] In some embodiments, the first dimerizing domain comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ IDNO: 425. In some embodiments, the first dimerizing domain has a sequence of SEQ ID NO: 425. In some embodiments, the second dimerizing domain comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 431. In some embodiments, the second dimerizing domain has a sequence of SEQ ID NO: 431.

[0584] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain and the first dimerizing domain; and the second polypeptide comprises, from N terminus to C terminus, the TCR beta variable domain and the second dimerizing domain. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, a first peptide linker, and the first dimerizing domain; and the second polypeptide comprises, from N terminus to C terminus, the TCR beta variable domain, a second peptide linker, and the second dimerizing domain. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain and the TCR alpha variable domain, and the second polypeptide comprises, from N terminus to C terminus, the second dimerizing domain and the TCR beta variable domain. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain, a first peptide linker, and the TCR alpha variable domain, and the second polypeptide comprises, from N terminus to C terminus, the second dimerizing domain, a second peptide linker, and the TCR beta variable domain.

[0585] In some embodiments, the first dimerizing domain comprises a TCR alpha constant (TRAC) domain or portion thereof. In some embodiments, the second dimerizing domain comprises a TCR beta constant (TRBC) domain or portion thereof. In some embodiments, the first dimerizing domain comprises a TCR alpha constant (TRAC) domain or portion thereof and the second dimerizing domain comprises a TCR beta constant (TRBC) domain or portion thereof.

[0586] In some embodiments, the first dimerizing domain and the second dimerizing domain are linked. In some embodiments, the first dimerizing domain and the second dimerizing domain are linked by a single disulfide bridge. In some embodiments, the first dimerizing domain and the second dimerizing domain are linked by two or more disulfide bridges.

[0587] In some embodiments, the first polypeptide chain comprises the at least one tag. In some embodiments, the first polypeptide chain comprises from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, a linker, and the at least one tag. Insome embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the TCR alpha variable domain, and the first dimerizing domain. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, a linker, the TCR alpha variable domain, and the first dimerizing domain. In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10 or more. In some embodiments, the integer is from 1 to 10. In some embodiments, the integer is 1. In some embodiments, the integer is 2. In some embodiments, the integer is 3. In some embodiments, the integer is 4. In some embodiments, the integer is 5. In some embodiments, the integer is 6. In some embodiments, the integer is 7. In some embodiments, the integer is 8. In some embodiments, the integer is 9. In some embodiments, the integer is 10.

[0588] In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain, the TCR alpha variable domain, and the at least one tag. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the first dimerizing domain, and the TCR alpha variable domain.

[0589] In some embodiments, the second polypeptide chain comprises the at least one tag. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the TCR beta variable domain, and the second dimerizing domain. In some embodiments, wherein the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, a linker, the TCR beta variable domain, and the second dimerizing domain. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, and the at least one tag. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, a linker, and the at least one tag. In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10 or more. In some embodiments, the integer is from 1 to 10. In some embodiments, the integer is 1. In some embodiments, the integer is 2. In some embodiments, the integer is 3. In some embodiments, the integer is 4. In some embodiments, the integer is 5. In some embodiments, the integer is 6. In some embodiments, the integer is 7. In some embodiments, the integer is 8. In some embodiments, the integer is 9. In some embodiments, the integer is 10.

[0590] In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the second dimerizing domain, the TCR alpha beta variable domain, and the at least one tag. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the second dimerizing domain, and the TCR beta variable domain.

[0591] In some embodiments, the at least one tag comprises two or more tags. In some embodiments, the at least one tag comprises two tags. In some embodiments, the at least one tag comprises three tags. In some embodiments, the at least one tag comprises four tags. In some embodiments, the at least one tag comprises five tags. In some embodiments, each of the two or more tags comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 126. In some embodiments, the at least one tag comprises a sequence of SEQ ID NO: 126. In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10 or more. In some embodiments, the integer is from 1 to 10. In some embodiments, the integer is 1. In some embodiments, the integer is 2. In some embodiments, the integer is 3. In some embodiments, the integer is 4. In some embodiments, the integer is 5. In some embodiments, the integer is 6. In some embodiments, the integer is 7. In some embodiments, the integer is 8. In some embodiments, the integer is 9. In some embodiments, the integer is 10.

[0592] In some embodiments, the at least one tag comprises two tags. In some embodiments a sTCR comprising two tags results in killing of target cells at a peptide concentration less than IxlO'6M, less than 5xl0'7M, less than IxlO'7M, less than 5xl0'8M, or less than IxlO'8M, less than 5xl0'9M, or less than IxlO'9M. In some embodiments a sTCR comprising two tags results in Ki-67 expression at a peptide concentration less than IxlO'6M, less than 5xl0'7M, less than IxlO'7M, less than 5xl0'8M, or less than IxlO'8M, less than 5xl0'9M, or less than IxlO'9M. In some embodiments a sTCR comprising two tags results in Granzyme B production at a peptide concentration less than IxlO'6M, less than 5xl0'7M, less than IxlO'7M, less than 5xl0'8M, or less than IxlO'8M, less than 5xl0'9M, or less than IxlO'9M. In some embodiments, a sTCR comprising two tags results in a 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or more increase in percent of target cells killed relative to an otherwise identical sTCR comprising one tag. In some embodiments, a sTCR comprising two tags results in a 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or more increase in Ki -67 expression relative to an otherwise identical sTCR comprising one tag. In some embodiments, a sTCR comprising two tags results in a 5-fold,10-fold, 15-fold, 20-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or more increase in Granzyme B production relative to an otherwise identical sTCR comprising one tag.

[0593] In some embodiments, the at least one tag comprises three tags. In some embodiments a sTCR comprising three tags results in killing of target cells at a peptide concentration less than IxlO'6M, less than 5xl0'7M, less than IxlO'7M, less than 5xl0'8M, or less than IxlO'8M, less than 5xl0'9M, or less than IxlO'9M. In some embodiments a sTCR comprising three tags results in Ki-67 expression at a peptide concentration less than IxlO'6M, less than 5xl0'7M, less than IxlO'7M, less than 5xl0'8M, or less than IxlO'8M, less than 5xl0'9M, or less than IxlO'9M. In some embodiments a sTCR comprising three tags results in Granzyme B production at a peptide concentration less than IxlO'6M, less than 5xl0'7M, less than IxlO'7M, less than 5xl0'8M, or less than IxlO'8M, less than 5xl0'9M, or less than IxlO'9M. In some embodiments, a sTCR comprising three tags results in a 5- fold, 10-fold, 15-fold, 20-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or more increase in percent of target cells killed relative to an otherwise identical sTCR comprising two tags. In some embodiments, a sTCR comprising three tags results in a 5-fold, 10-fold, 15-fold, 20- fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or more increase in Ki-67 expression relative to an otherwise identical sTCR comprising two tags. In some embodiments, a sTCR comprising three tags results in a 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold or more increase in Granzyme B production relative to an otherwise identical sTCR comprising two tags.

[0594] In some embodiments, the two or more tags are linked to the same first polypeptide chain. In some embodiments, the two or more tags are linked to the same second polypeptide chain. In some embodiments, the two or more tags are linked to different polypeptide chains. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, and the two or more tags operably linked. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, a first linker, a first tag, a second linker, and a second tag. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, the two or more tags operably linked, the TCR alpha variable domain, and the first dimerizing domain. In some embodiments, the first polypeptide chain comprises, from N terminus to C terminus, a first tag, a first linker, a second tag, a second linker, the TCR alpha variable domain, and the first dimerizing domain. In some embodiments, the second polypeptide chain comprises, from Nterminus to C terminus, the TCR beta variable domain, the second dimerizing domain, and the two or more tags operably linked. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, a first linker, a first tag, a second linker, and a second tag. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, the two or more tags operably linked, the TCR beta variable domain, and the second dimerizing domain. In some embodiments, the second polypeptide chain comprises, from N terminus to C terminus, a first tag, a first linker, a second tag, a second linker, the TCR beta variable domain, and the second dimerizing domain. In some embodiments, the first tag and the second tag comprise the same sequence. In some embodiments, the sequence comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 126. In some embodiments, the at least one tag comprises a sequence of SEQ ID NO: 126. In some embodiments, the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10 or more. In some embodiments, the integer is from 1 to 10. In some embodiments, the integer is 1. In some embodiments, the integer is 2. In some embodiments, the integer is 3. In some embodiments, the integer is 4. In some embodiments, the integer is 5. In some embodiments, the integer is 6. In some embodiments, the integer is 7. In some embodiments, the integer is 8. In some embodiments, the integer is 9. In some embodiments, the integer is 10.

[0595] In some embodiments, the soluble TCR comprises two or more soluble TCRs. In some embodiments, each soluble TCR of the two or more soluble TCRs recognizes a different peptide:MHC complex. In some embodiments, the different peptide:MHC complexes are on the same target cell. In some embodiments, the different peptide:MHC complexes are on different target cells. In some embodiments, the different peptide:MHC complexes comprise different epitope sequences. In some embodiments, the different peptide:MHC complexes comprise different MHC molecules encoded by different HLA alleles. In some embodiments, the different peptide:MHC complexes comprise different epitope sequences presented by the same MHC molecule encoded by the same HLA allele. In some embodiments, the different peptide:MHC complexes comprise different epitope sequences presented by different MHC molecules encoded by different HLA alleles. In some embodiments, the different peptide:MHC complexes comprise the same epitope sequence presented by different MHC molecule encoded by different HLA alleles.

[0596] In some embodiments, the soluble TCR comprises a TCR beta variable domain comprising a sequence comprising a complementarity determining region 3 (CDR3). In some embodiments, the CDR3 comprises an amino acid with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 430. In some embodiments, CDR3 comprises an amino acid sequence of SEQ ID NO: 430. In some embodiments, the CDR3 comprises an amino acid with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 435. In some embodiments, CDR3 comprises an amino acid sequence of SEQ ID NO: 435. In some embodiments, the CDR3 comprises an amino acid with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 445. In some embodiments, CDR3 comprises an amino acid sequence of SEQ ID NO: 445. In some embodiments, the CDR3 comprises an amino acid with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 456. In some embodiments, CDR3 comprises an amino acid sequence of SEQ ID NO: 456. In some embodiments, the TCR beta variable domain comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 327. In some embodiments, TCR beta variable domain comprises an amino acid sequence of SEQ ID NO: 327. In some embodiments, the TCR beta variable domain comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 434. In some embodiments, TCR beta variable domain comprises an amino acid sequence of SEQ ID NO: 434. In some embodiments, the TCR beta variable domain comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 442. In some embodiments, TCR beta variable domain comprises an amino acid sequence of SEQ ID NO: 442. In some embodiments, the TCR beta variable domain comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 453. In some embodiments, TCR beta variable domain comprises an amino acid sequence of SEQ ID NO: 453. In some embodiments, the TCR beta chain construct comprises a CDR1 having an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 428. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO: 428. In some embodiments, the TCR beta chain constructcomprises a CDR1 having an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 443. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO: 443. In some embodiments, the TCR beta chain construct comprises a CDR2 having an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 429. In some embodiments, CDR2 comprises an amino acid sequence of SEQ ID NO: 429. In some embodiments, the TCR beta chain construct comprises a CDR2 having an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 444. In some embodiments, CDR2 comprises an amino acid sequence of SEQ ID NO: 444.

[0597] In some embodiments, the soluble TCR comprises the TCR alpha variable domain comprising a CDR1, a CDR2, and a CDR3. In some embodiments, the CDR1 has an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 422. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO: 422. In some embodiments, the CDR1 has an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 438. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO: 438. In some embodiments, the CDR1 has an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 448. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO: 448. In some embodiments, the CDR2 comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 423. In some embodiments, the CDR2 comprises a sequence of SEQ ID NO: 423. In some embodiments, the CDR2 comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 239. In some embodiments, the CDR2 comprises a sequence of SEQ ID NO: 239. In some embodiments, the CDR2 comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 449. In some embodiments, the CDR2 comprises a sequence of SEQ ID NO: 449. In some embodiments, the CDR3 comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 424. In some embodiments, the CDR3 comprises a sequence of SEQ ID NO: 424. In some embodiments, the CDR3 comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence ofSEQ ID NO: 440. In some embodiments, the CDR3 comprises a sequence of SEQ ID NO: 440. In some embodiments, the CDR3 comprises a sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 450. In some embodiments, the CDR3 comprises a sequence of SEQ ID NO: 450. In some embodiments, the TCR alpha variable domain comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 421. In some embodiments, TCR alpha variable domain comprises an amino acid sequence of SEQ ID NO: 421. In some embodiments, the TCR alpha variable domain comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 437. In some embodiments, TCR alpha variable domain comprises an amino acid sequence of SEQ ID NO: 437. In some embodiments, the TCR alpha variable domain comprises an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence of SEQ ID NO: 447. In some embodiments, TCR alpha variable domain comprises an amino acid sequence of SEQ ID NO: 447.

[0598] In some embodiments, the soluble TCR comprises (a) the TCR beta variable domain having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to sequence of SEQ ID NO: 427, the TCR beta variable domain having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to sequence of SEQ ID NO: 434, the TCR beta variable domain having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to sequence of SEQ ID NO: 442, or the TCR beta variable domain having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to sequence of SEQ ID NO: 456 and (b) the TCR alpha variable domain having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to sequence of SEQ ID NO: 421, the TCR alpha variable domain having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to sequence of SEQ ID NO: 437, or the TCR alpha variable domain having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to sequence of SEQ ID NO: 447.

[0599] In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 199. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%,at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 200. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 201. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 202. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 203. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 204. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 205. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 359. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 432. In some embodiments, the soluble TCR binds to a peptide:MHC complex having an epitope sequence with at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the epitope is in complex with an MHC molecule encoded by an HLA allele. In some embodiments, the HLA allele is HLA-A*02:01. In some embodiments, the HLA allele is HLA-C*08:02.

[0600] In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 121, 127, 130, 534, 134, 136, 138, and 533. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 121. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 127. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 130. In someembodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 534. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 134. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 136. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 138. In some embodiments, the tag-sTCR comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 533.

[0601] In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 141-148 and 459-475. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 141. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 142. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 143. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 144. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 145. In some embodiments, the polynucleotide encoding the sTCR comprises a sequence with at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence of SEQ ID NO: 146. In some embodiments, the po...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A composition comprising a soluble T-cell receptor (TCR) having at least one tag (tag- sTCR), or a polynucleotide encoding the tag-sTCR, wherein the tag-sTCR binds to a peptide:MHC complex of a target cell.

2. The composition of claim 1, wherein the composition further comprises a chimeric receptor (CAR) or polynucleotide encoding the CAR, and wherein the CAR comprises (i) an extracellular domain comprising an anti-tag binding domain that binds to the at least one tag of the tag-sTCR, (ii) a transmembrane domain, and (iii) an intracellular domain.

3. The composition of claim 2, wherein the CAR is expressed on a surface of an immune cell.

4. A composition comprising: a soluble TCR having at least one tag (tag-sTCR) or a polynucleotide encoding the tag- sTCR; and an immune cell comprising a chimeric receptor (CAR) or polynucleotide encoding the CAR, wherein the CAR comprises (i) an extracellular domain comprising an anti-tag binding domain that binds to the at least one tag of the tag-sTCR, (ii) a transmembrane domain, and (iii) an intracellular domain.

5. The composition of claim 4, wherein the tag-sTCR binds to a peptide:MHC complex of a target cell.

6. The composition of any one of claims 1-5, wherein the soluble TCR comprises a peptide:MHC complex engager comprising a TCR alpha variable domain and a TCR beta variable domain.

7. The composition of any one of claims 1-6, wherein the soluble TCR is not a single chain TCR.

8. The composition of any one of claims 1-7, wherein the soluble TCR does not comprise a constant region of a TCR.

9. The composition of any one of claims 1-5 and 8, wherein the soluble TCR is a single chain TCR, and wherein the TCR alpha variable domain is operably linked to the TCR beta variable domain.

10. The composition of claim 9, wherein the soluble TCR comprises, from N-terminus to C-terminus, the TCR alpha variable domain operably linked to the TCR beta variable domain.

11. The composition of claim 9, wherein the soluble TCR comprises, from N-terminus to C-terminus, the TCR beta variable domain operably linked to the TCR alpha variable domain.

12. The composition of any one of claims 9-11, wherein the tag-sTCR is a fusion protein, and wherein the at least one tag is operably linked to the soluble TCR.

13. The composition of claim 12, wherein the at least one tag is operably linked to the N- terminus of the soluble TCR.

14. The composition of claim 12, wherein the at least one tag is operably linked to the C- terminus of the soluble TCR.

15. The composition of any one of claims 1-5 and 8, wherein the soluble TCR is a single chain TCR, and wherein the TCR comprises, from N-terminus to C-terminus, the TCR beta variable domain, the TCR beta constant domain, the TCR alpha variable domain, and the TCR alpha constant domain.

16. The composition of claim 15, wherein the at least one tag is operably linked to the N- terminus of the single chain TCR.

17. The composition of claim 15 or 16, wherein the soluble TCR comprises a linker.

18. The composition of claim 17, wherein the linker is operably linked to the TCR alpha constant domain and the TCR alpha variable domain.

19. The composition of claim 17 or 18, wherein the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

20. The composition of claim 19, wherein n is 7.

21. The composition of any one of claims 17-20, wherein the soluble TCR comprises an additional tag operably linked to the C-terminus of the soluble TCR.

22. The composition of claim 21, wherein the additional tag is a lOXHIS-sortag operably linked to the C-terminus of the soluble TCR.

23. The composition of claim 21 or 22, wherein the soluble TCR comprises, from N- terminus to C-terminus, the at least one tag, the TCR beta variable domain, the TCR beta constant domain, the linker, the TCR alpha variable domain, the TCR alpha constant domain, and the additional tag.

24. The composition of any one of claims 1-5 and 8, wherein the soluble TCR is a single chain TCR, and wherein the TCR comprises, from N-terminus to C-terminus, the TCR alpha variable domain, the TCR alpha constant domain, the TCR beta variable domain, and the TCR beta constant domain.

25. The composition of claim 24, wherein the at least one tag is operably linked to the TCR beta variable domain.

26. The composition of claim 24 or 25, wherein the soluble TCR comprises a linker.

27. The composition of claim 26, wherein the linker is operably linked to the at least one tag and the TCR beta constant domain.

28. The composition of claim 26 or 27, wherein the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

29. The composition of claim 28, wherein n is 4.

30. The composition of any one of claims 26-29, wherein the soluble TCR comprises an additional tag operably linked to the C-terminus of the soluble TCR.

31. The composition of claim 30, wherein the additional tag is a lOXHIS-sortag operably linked to the C-terminus of the soluble TCR.

32. The composition of claim 30 or 31, wherein the soluble TCR comprises, from N- terminus to C-terminus, the TCR alpha variable domain, the TCR alpha constant domain, the linker, the at least one tag, the TCR beta variable region, the TCR beta constant region, and the additional tag.

33. The composition of any one of claims 15-30, wherein the soluble TCR comprises one or more disulfide bridges between the TCR alpha constant domain and the TCR beta constant domain.

34. The composition of any one of claims 15-33, wherein the soluble TCR comprises a modification comprising removal of one or more N-glycosylation sites in the TCR alpha constant domain and / or TCR beta constant domain.

35. The composition of any one of claims 15-34, wherein the TCR alpha variable domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR alpha variable domain sequences of Tables 5, 6, 8, 11, and 12.

36. The composition of claim 35, wherein the TCR beta variable domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR beta variable domain sequences of Tables 5, 6, 8, 11, and 12.

37. The composition of claim 35 or 36, wherein the TCR alpha constant domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR alpha constant domain sequences of Tables 5, 6, 8, 11, and 12.

38. The composition of claim 37, wherein the TCR beta constant domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR beta constant domain sequences of Tables 5, 6, 8, 11, and 12.

39. The composition of any one of claims 30-38, wherein the additional tag comprises a sequence of SEQ ID NO : 131.

40. The composition of any one of claims 2-39, wherein the at least one tag binds to the anti-tag binding domain of the CAR.

41. The composition of any one of claims 2-40, wherein the anti -tag binding domain is an antibody or antigen binding fragment thereof.

42. The composition of any one of claims 1-41, wherein the at least one tag is not an affinity tag.

43. The composition of any one of claims 1-42, wherein the at least one tag does not comprise a fluorophore.

44. The composition of any one of claims 1-43, wherein the tag-sTCR does not comprise an antibody or any fragment thereof.

45. The composition of any one of claims 1-44, wherein the tag-sTCR does not comprise an scFv.

46. The composition of any one of claims 1-44, wherein the at least one tag is a polypeptide that is absent in eukaryotic systems.

47. The composition of any one of claims 1-46, wherein the at least one tag comprises less than 200, less than 100, less than 50, less than 30 or less than 20 amino acids.

48. The composition of any one of claims 1-47, wherein the at least one tag comprises at least 5, at least 10, at least 15, at least 20, at least 50, at least 100 or more amino acids.

49. The composition of any one of claims 1-48, wherein the at least one tag does not comprise modified amino acids or unnatural amino acids.

50. The composition of any one of claims 1-49, wherein all amino acids of the at least one tag are non-modified amino acids with natural peptide bonds.

51. The composition of any one of claims 1-50, wherein the at least one tag forms a stable alpha-helical structure.

52. The composition of any one of claims 1-51, wherein the at least one tag is hydrophilic and neutral at physiological pH.

53. The composition of any one of claims 1-52, wherein the at least one tag comprises a sequence of SEQ ID NO: 126, or a sequence that comprises less than 3 amino acids differing from the above within the sequence.

54. The composition of claim 53, wherein the at least one tag further comprises 1, 2, 3, 4 or more amino acids on the N terminus of the sequence, or the C terminus of the sequence, or both the N terminus and the C terminus of the sequence.

55. The composition of any one of claims 1-54, wherein the at least one tag is recognized by the anti -tag binding domain of the CAR with a Ka of less than 1,000 nM, less than 500 nM, less than 250 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM or less than 10 nM.

56. The composition of any one of claims 6-55, wherein the peptideMHC complex engager specifically binds to an endogenous disease specific antigen.

57. The composition of any one of claims 6-56, wherein the peptideMHC complex engager specifically binds to a cancer antigen.

58. The composition of claim 57, wherein the cancer antigen is a neoantigen, a tumor associated antigen, or a viral antigen.

59. The composition of any one of claims 6-58, wherein the peptideMHC complex engager binds to the peptideMHC complex comprising an epitope from the endogenous disease specific antigen that is presented on the surface of the target cell.

60. The composition of claim 59, wherein the target cell is a cancer cell.

61. The composition of claim 59 or 60, wherein the peptide:MHC complex engager binds to the peptideMHC complex with a Ka of less than 1,000 nM, less than 500 nM, less than 250 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM or less than 10 nM.

62. The composition of any one of claims 1-3 and 5-61, wherein an MHC of the peptide:MHC complex is a class I MHC.

63. The composition of any one of claims 1-3 and 5-61, wherein an MHC of the peptide:MHC complex is a class II MHC.

64. The composition of any one of claims 59-63, wherein the epitope is derived from a RAS polypeptide or a fragment thereof.

65. The composition of claim 64, wherein the RAS polypeptide is a mutated RAS polypeptide.

66. The composition of claim 64 or 65, wherein the epitope derived from the RAS polypeptide or a fragment thereof comprises a G12C, G12V, G12D, or G12R mutation.

67. The composition of any one of claims 64-66, wherein the peptide:MHC complex comprises an epitope having a sequence selected from the group consisting of SEQ ID NOs: 199-205, 359, and 432.

68. The composition of claim 66, wherein the peptide:MHC complex comprises an epitope having a sequence of SEQ ID NOs: 202 or 432.

69. The composition of any one of claims 64-68, wherein an MHC of the peptide:MHC complex comprising the epitope derived from the RAS polypeptide or a fragment thereof comprises an MHC encoded by an HLA-A*02:01 allele or an HLA-C*08:02 allele.

70. The composition of any one of claims 59-63, wherein the epitope is derived from a GATA3 polypeptide or a fragment thereof.

71. The composition of claim 70, wherein the GATA3 polypeptide is a mutated GATA3 polypeptide.

72. The composition of claim 70 or 71, wherein the GAT A3 polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 485-487.

73. The composition of any one of claims 70-72, wherein the epitope derived from the GATA3 polypeptide or a fragment thereof comprises the epitope having a sequence selected from the group consisting of an epitope from SEQ ID NO: 485, an epitope from SEQ ID NO: 486, and an epitope from SEQ ID NO: 487.

74. The composition of claim 72, wherein the epitope comprises a sequence MLTGPPARV (SEQ ID NO: 15).

75. The composition of any one of claims 59-63, wherein the epitope is derived from a Human Papillomavirus (HPV) protein.

76. The composition of claim 75, wherein the HPV protein is a HPV16 E7 protein.

77. The composition of claim 76, wherein the epitope comprises a sequence of SEQ ID NO: 458.

78. The composition of any one of claims 9-77, wherein the single chain TCR comprises a linker between the TCR alpha variable domain and the TCR beta variable domain.

79. The composition of claim 78, wherein the linker is a polypeptide linker.

80. The composition of claim 78 or 79, wherein the linker comprises the at least one tag sequence.

81. The composition of any one of claims 78-80, wherein the linker comprises a sequence selected from the group consisting of SEQ ID NOs: 125, 128, 135, and 488.

82. The composition of any one of claims 9-81, wherein the soluble TCR is a single chain TCR, and wherein the single chain TCR comprises, from N terminus to C terminus, the TCR alpha variable domain, the TCR alpha constant domain, a first linker, a first tag, a second linker, a second tag, the TCR beta variable domain, and the TCR beta constant domain.

83. The composition of claim 82, wherein the first tag or the second tag comprises a sequence of SEQ ID NO: 126.

84. The composition of claim 82, wherein the first tag and the second tag each comprises a sequence of SEQ ID NO: 126.

85. The composition of any one of claims 82-84, wherein the first linker or the second linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

86. The composition of claim 85, wherein n is 2 or 4.

87. The composition of any one of claims 82-86, wherein the first linker comprises a sequence of (GGGGS)4, and the second linker comprises a sequence of GGGGS.

88. The composition of any one of claims 82-86, wherein the first linker comprises a sequence of (GGGGS)2, and the second linker comprises a sequence of GGGGS.

89. The composition of any one of claims 82-88, wherein the soluble TCR comprises a sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 552, 556, and 557.

90. The composition of any one of claims 6-8 and 40-81, wherein the TCR alpha variable domain and the TCR beta variable domain are on two separate polypeptide chains comprising a first polypeptide chain and a second polypeptide chain.

91. The composition of claim 90, wherein the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 138, 541, 543, 544, 545, 547, 548, and 550.

92. The composition of claim 90 or 91, wherein the first polypeptide chain comprises a first dimerizing domain fused to the TCR alpha variable domain, and the secondpolypeptide chain comprises a second dimerizing domain fused to the TCR beta variable domain.

93. The composition of claim 92, wherein the first dimerizing domain or the second dimerizing domain comprises at least 2, at least 5, at least 10 or more amino acids of a TCR extracellular domain adjacent to a TCR transmembrane domain.

94. The composition of claim 92 or 93, wherein the first dimerizing domain comprises a sequence as set forth in SEQ ID NO: 425.

95. The composition of any one of claims 92-94, wherein the second dimerizing domain comprises a sequence as set forth in SEQ ID NO: 431.

96. The composition of any one of claims 90-94, wherein the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain and the first dimerizing domain; and the second polypeptide comprises, from N terminus to C terminus, the TCR beta variable domain and the second dimerizing domain.

97. The composition of any one of claims 90-96, wherein the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, a first peptide linker, and the first dimerizing domain; and the second polypeptide comprises, from N terminus to C terminus, the TCR beta variable domain, a second peptide linker, and the second dimerizing domain.

98. The composition of any one of claims 90-94, wherein the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain and the TCR alpha variable domain, and the second polypeptide comprises, from N terminus to C terminus, the second dimerizing domain and the TCR beta variable domain.

99. The composition of any one of claims 90-94 and 98, wherein the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain, a first peptide linker, and the TCR alpha variable domain, and the second polypeptide comprises, from N terminus to C terminus, the second dimerizing domain, a second peptide linker, and the TCR beta variable domain.

100. The composition of any one of claims 92-99, wherein the first dimerizing domain comprises a TCR alpha constant (TRAC) domain or portion thereof; and the second dimerizing domain comprises a TCR beta constant (TRBC) domain or portion thereof.

101. The composition of any one of claims 92-100, wherein the first dimerizing domain and the second dimerizing domain are linked by one or more disulfide bridges.

102. The composition of claim 101, wherein the first dimerizing domain and the second dimerizing domain are linked by a single disulfide bridge.

103. The composition of any one of claims 90-102, wherein the first polypeptide chain comprises the at least one tag.

104. The composition of claim 103, wherein the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, and the at least one tag.

105. The composition of claim 104, wherein the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, a linker, and the at least one tag.

106. The composition of claim 103, wherein the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the TCR alpha variable domain, and the first dimerizing domain.

107. The composition of claim 106, wherein the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, a linker, the TCR alpha variable domain, and the first dimerizing domain.

108. The composition of claim 105 or 107, wherein the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

109. The composition of claim 103, wherein the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain, the TCR alpha variable domain, and the at least one tag.

110. The composition of claim 103, wherein the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the first dimerizing domain, and the TCR alpha variable domain.

111. The composition of any one of claims 90-102, wherein the second polypeptide chain comprises the at least one tag.

112. The composition of claim 111, wherein the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the TCR beta variable domain, and the second dimerizing domain.

113. The composition of claim 112, wherein the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, a linker, the TCR beta variable domain, and the second dimerizing domain.

114. The composition of claim 111, wherein the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, and the at least one tag.

115. The composition of claim 114, wherein the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, a linker, and the at least one tag.

116. The composition of claim 113 or 115, wherein the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

117. The composition of claim 111, wherein the second polypeptide chain comprises, from N terminus to C terminus, the second dimerizing domain, the TCR beta variable domain, and the at least one tag.

118. The composition of claim 111, wherein the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the second dimerizing domain, and the TCR beta variable domain.

119. The composition of any one of claims 1-118, wherein the at least one tag comprises two or more tags.

120. The composition of claim 119, wherein the at least one tag comprises two tags.

121. The composition of claim 119, wherein the at least one tag comprises three tags.

122. The composition of any one of claims 119-121, wherein each tag of the two or more tags comprises a sequence of SEQ ID NO: 126.

123. The composition of any one of claims 119-122, wherein the two or more tags are operably linked by a linker having a sequence of (GGGGS)n, where n is an integer from 1 to 10.

124. The composition of claim 123, wherein the two or more tags are operably linked by a linker having a sequence of (GGGGS)4.

125. The composition of any one of claims 119-123, wherein the two or more tags are linked to the same first polypeptide chain or the same second polypeptide chain.

126. The composition of any one of claims 119-123, wherein the two or more tags are linked to different polypeptide chains.

127. The composition of any one of claims 119-126, wherein the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, and the two or more tags operably linked.

128. The composition of claim 127, wherein the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, a first linker, a first tag, a second linker, and a second tag.

129. The composition of any one of claims 119-126, wherein the first polypeptide chain comprises, from N terminus to C terminus, the two or more tags operably linked, the TCR alpha variable domain, and the first dimerizing domain.

130. The composition of claim 129, wherein the first polypeptide chain comprises, from N terminus to C terminus, a first tag, a first linker, a second tag, a second linker, the TCR alpha variable domain, and the first dimerizing domain.

131. The composition of any one of claims 119-126, wherein the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, and the two or more tags operably linked.

132. The composition of claim 131, wherein the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, a first linker, a first tag, a second linker, and a second tag.

133. The composition of any one of claims 119-126, wherein the second polypeptide chain comprises, from N terminus to C terminus, the two or more tags operably linked, the TCR beta variable domain, and the second dimerizing domain.

134. The composition of claim 133, wherein the second polypeptide chain comprises, from N terminus to C terminus, a first tag, a first linker, a second tag, a second linker, the TCR beta variable domain, and the second dimerizing domain.

135. The composition of any one of claims 128, 130, 132 and 134, wherein the first tag and the second tag comprise the same sequence.

136. The composition of claim 135, wherein the sequence comprises a sequence of SEQ ID NO: 126.

137. The composition of any one of claims 128, 130, 132 and 134-136, wherein the first linker or the second linker comprises a sequence of (GGGGS)n, where n is an integer from 1 to 10.

138. The composition of any one of claims 1-137, wherein the soluble TCR comprises two or more soluble TCRs, wherein each soluble TCR of the two or more soluble TCRs recognizes a different peptide:MHC complex, wherein (i) the different peptide:MHC complexes are on the same target cell or different target cells, (ii) the different peptide:MHC complexes comprise different epitope sequences, and / or (iii) thedifferent peptide:MHC complexes comprise different MHC molecules encoded by different HLA alleles.

139. The composition of claim 138, wherein the different peptide:MHC complexes comprise different epitope sequences presented by the same MHC molecule encoded by the same HLA allele.

140. The composition of claim 138 or 139, wherein the different peptide:MHC complexes comprise different epitope sequences presented by different MHC molecules encoded by different HLA alleles.

141. The composition of any one of claims 138-140, wherein the different peptide:MHC complexes comprise the same epitope sequence presented by different MHC molecule encoded by different HLA alleles.

142. The composition of any one of claims 138-141, wherein the different peptide:MHC complexes are on the same target cell.

143. The composition of any one of claims 138-141, wherein the different peptide:MHC complexes are on different target cells.

144. The composition of any one of claims 1-143, wherein the soluble TCR comprises the TCR beta variable domain comprising a complementarity determining region 3 (CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 430, 435, 445, and 456.

145. The composition of any one of claims 1-144, wherein the TCR beta variable domain comprises an amino acid sequence with at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 427, 434, 442, and 453.

146. The composition of any one of claims 1-145, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence as set forth in SEQ ID NOs: 428 or 443 and a complementarity determining region 2 (CDR2) having an amino acid sequence as set forth in SEQ ID NO. 429 or 444.

147. The composition of any one of claims 1-146, wherein the soluble TCR comprises the TCR alpha variable domain comprising a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 422, 438, and 448, the CDR2 has an amino acid sequence selected from the-m-group consisting of SEQ ID NOs: 423, 239, and 449, and the CDR3 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 424, 440, and 450.

148. The composition of any one of claims 1-147, wherein the TCR alpha variable domain comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 421, 437, and 447.

149. The composition of any one of claims 1-148, wherein the soluble TCR comprises (a) the TCR beta variable domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 427, 434, 442, and 453, or an amino acid sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 427, 434, 442, and 453, and (b) the TCR alpha variable domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 421, 437, and 447, or an amino acid sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 421, 437, and 447.

150. The composition of any one of claims 1-149, wherein the soluble TCR binds to the peptide:MHC complex having an epitope sequence selected from the group consisting of SEQ ID NOs: 199-205, 359, 432, and 15 in complex with an MHC molecule encoded by an HLA-A*02:01 allele or an HLA-C*08:02 allele.

151. The composition of any one of claims 1-150, wherein the tag-sTCR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 121, 127, 130, 534, and 134.

152. The composition of any one of claims 1-150, wherein the polynucleotide encoding the tag-sTCR comprises a sequence selected from the group consisting of SEQ ID NOs: 141-148 and 459-475.

153. A cell comprising a chimeric antigen receptor (CAR) or a polynucleotide encoding the CAR, wherein the CAR comprises (i) an extracellular domain comprising a domain that binds to an ALFA tag, (ii) a transmembrane domain, and (iii) an intracellular domain, and wherein the anti -tag binding domain comprises a CDR1 region comprising an amino acid sequence of SEQ ID NO: 477, a CDR2 region comprising an amino acid sequence of SEQ ID NO: 478, and a CDR3 region comprising an amino acid sequence of SEQ ID NO: 479.

154. The cell of claim 153, wherein the transmembrane domain is derived from CD28, CD3E, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86,CD134, CD 154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD 18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL17Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD 103, ITGAL, CD 11 a, LFA-1, ITGAM, CD 11b, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or NKG2C or a functional variant thereof.

155. The cell of claim 153 or 154, wherein the transmembrane domain comprises a CD8a transmembrane domain.

156. The cell of any one of claims 153-155, wherein the extracellular domain is operably linked to the transmembrane domain by a hinge domain.

157. The cell of claim 156, wherein the hinge domain is a CD8a hinge domain.

158. The cell of any one of claims 153-157, wherein the intracellular domain comprises a primary cytoplasmic signaling sequence derived from CD3 combined with a costimulatory signaling region.

159. The cell of claim 158, wherein the costimulatory signaling region is derived from CD28, 0X40, 4- IBB, ICOS or a functional variant thereof.

160. The cell of any one of claims 153-159, wherein the extracellular domain comprises an antibody, a functional antibody fragment, a single chain variable fragment (scFv), an Fab, a single-domain antibody (sdAb), a nanobody, a VH domain, a VL domain, a VNAR domain, a VHH domain, a bispecific antibody, a diabody, or a functional fragment or a combination thereof.

161. The cell of any one of claims 153-160, wherein the transmembrane domain is comprises a CD8a transmembrane domain and wherein the CD8a transmembrane domain has a sequence of SEQ ID NO: 480.

162. The cell of claim 161, wherein the extracellular domain is operably linked to the transmembrane domain by a hinge domain and wherein the hinge domain is a CD8a hinge domain having a sequence of SEQ ID NO: 490.

163. The cell of any one of claims 153-160, wherein the intracellular domain comprises a primary cytoplasmic signaling sequence derived from CD3 having a sequence of SEQID NO: 482 combined with a costimulatory signaling region derived from 4-1BB having a sequence of SEQ ID NO: 481.

164. The cell of any one of claims 153-163, wherein the CAR comprises a sequence with at least 85% identity to SEQ ID NO: 476.

165. The cell of any one of claims 153-164, wherein the polynucleotide encoding the CAR comprises a sequence with at least 85% identity to SEQ ID NO: 484.

166. The cell of any one of claims 153-165, wherein the cell is a T cell.

167. The cell of claim 166, wherein the T cell is a CD4 T cell.

168. The cell of claim 166, wherein the T cell is a CD8 T cell.

169. A pharmaceutical composition comprising the tag-sTCR or the polynucleotide encoding the tag-sTCR of any one of claims 1-152 and a pharmaceutically acceptable carrier.

170. A pharmaceutical composition comprising the immune cell comprising the CAR or polynucleotide encoding the CAR of any one of claims 153-168 and a pharmaceutically acceptable carrier.

171. Use of the tag-sTCR or the polynucleotide encoding the tag-sTCR of any one of claims 1-152 or the immune cell comprising the CAR or polynucleotide encoding the CAR of any one of claims 153-168 in the manufacture of a medicament in treating a disease or a condition in a subject in need thereof.

172. The use of claim 171, wherein the disease or the condition is a cancer.

173. A method of treating a disease or condition in a subject in need thereof, comprising(a) administering a composition comprising the composition of any one of claims 1-152; or(b) administering a therapeutically effective population of immune cells comprising the cell of any one of claims 153-168; thereby treating the disease or condition in the subject.

174. A method of preventing a disease or condition in a subject in need thereof, comprising(a) administering a composition comprising the composition of any one of claims 1-152; or(b) administering a therapeutically effective population of immune cells comprising the cell of any one of claims 153-168; thereby treating the disease or condition in the subject.

175. A method of treating a disease or a condition in a subject in need thereof, comprising(a) administering a soluble TCR comprising a tag (tag-sTCR) or a polynucleotide encoding the tag-sTCR to a subject in need thereof, wherein the tag-sTCR binds to a target cell in the subject; and(b) administering a therapeutically-effective population of immune cells comprising a chimeric receptor (CAR) or polynucleotide encoding the CAR to the subject, wherein the CAR comprises: (i) an extracellular domain comprising an anti-tag binding domain that binds to the tag-sTCR, (ii) a transmembrane domain, and (iii) an intracellular domain comprising an intracellular signaling domain; thereby treating the disease or condition in the subject.

176. The method of claim 175, wherein administering in (a) is prior to, concurrently, or after administering in (b).

177. The method of any one of claims 173-176, wherein the disease is a cancer.

178. The method of claim 177, wherein the cancer is a solid cancer or a liquid cancer.

179. The method of claim 177, wherein the cancer is a cancer expressing or diagnosed as expressing a tumor-associated antigen (TAA).

180. The method of any one of claims 175-178, wherein the transmembrane domain is derived from CD28, CD3s, CD45, CD4, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2Rbeta, IL2R gamma, IL17Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or NKG2C or a functional variant thereof.

181. The method of any one of claims 175-180, wherein the transmembrane domain comprises a CD8a transmembrane domain.

182. The method of any one of claims 175-181, wherein the extracellular domain is operably linked to the transmembrane domain by a hinge domain.

183. The method of claim 182, wherein the hinge domain is a CD8a hinge domain.

184. The method of any one of claims 175-183, wherein the intracellular domain comprises an a primary cytoplasmic signaling sequence derived from CD3^ combined with a costimulatory signaling region.

185. The method of claim 184, wherein the costimulatory signaling region is derived from CD28, 0X40, 4- IBB, ICOS or a functional variant thereof.

186. The method of any one of claims 175-185, wherein the extracellular domain comprises an antibody, a functional antibody fragment, a single chain variable fragment (scFv), an Fab, a single-domain antibody (sdAb), a nanobody, a VH domain, a VL domain, a VNAR domain, a VHH domain, a bispecific antibody, a diabody, or a functional fragment or a combination thereof.

187. The method of any one of claims 175-186, wherein the soluble TCR comprises an peptide:MHC complex engager comprising a TCR alpha variable domain and a TCR beta variable domain.

188. The method of any one of claims 175-187, wherein the soluble TCR is not a single chain TCR.

189. The method of any one of claims 175-188, wherein the soluble TCR does not comprise a constant region of a TCR.

190. The method of any one of claims 175-189, wherein the at least one tag is operably linked to the soluble TCR.

191. The method of any one of claims 175-187, wherein the soluble TCR is a single chain TCR, and wherein the TCR alpha variable domain is operably linked to the TCR beta variable domain.

192. The method of claim 191, wherein the soluble TCR comprises, from N-terminus to C- terminus, the TCR alpha variable domain operably linked to the TCR beta variable domain.

193. The method of claim 191, wherein the soluble TCR comprises, from N-terminus to C- terminus, the TCR beta variable domain operably linked to the TCR alpha variable domain.

194. The method of any one of claims 190-193, wherein the at least one tag is operably linked to the N-terminus of the soluble TCR.

195. The method of any one of claims 190-193, wherein the at least one tag is operably linked to the C-terminus of the soluble TCR.

196. The method of claim 187, wherein the soluble TCR is a single chain TCR, and wherein the TCR comprises, from N-terminus to C-terminus, the TCR beta variable domain, a TCR beta constant domain, the TCR alpha variable domain, and a TCR alpha constant domain.

197. The method of claim 196, wherein the at least one tag is operably linked to the N- terminus of the soluble TCR.

198. The method of claim 197, wherein the at least one tag is an ALFA-tag.

199. The method of any one of claims 196-198, wherein the soluble TCR comprises a first linker.

200. The method of claim 199, wherein the at least one tag is operably linked to the TCR beta variable domain via the first linker.

201. The method of claim 199 or 200, wherein the first linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

202. The method of claim 201, wherein n is 1.

203. The method of any one of claims 196-202, wherein the soluble TCR comprises a second linker.

204. The method of claim 203, wherein the second linker is operably linked to the TCR beta constant domain and the TCR alpha variable domain.

205. The method of claim 203 or 204, wherein the second linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

206. The method of claim 205, wherein n is 7.

207. The method of any one of claims 193-206, wherein the soluble TCR comprises an additional tag operably linked to the C-terminus of the soluble TCR.

208. The method of claim 207, wherein the additional tag is a lOXHIS-sortag operably linked to the C-terminus of the soluble TCR.

209. The method of claim 207 or 208, wherein the soluble TCR comprises, from N-terminus to C-terminus, the at least one tag, the TCR beta variable domain, the TCR beta constant domain, the second linker, the TCR alpha variable domain, the TCR alpha constant domain, and the additional tag.

210. The method of claim 207 or 208, wherein the soluble TCR comprises, from N-terminus to C-terminus, the at least one tag, the first linker, the TCR beta variable domain, the TCR beta constant domain, the second linker, the TCR alpha variable domain, the TCR alpha constant domain, and the additional tag.

211. The method of claim 175, wherein the soluble TCR is a single chain TCR, and wherein the TCR comprises, from N-terminus to C-terminus, the TCR alpha variable domain, a TCR alpha constant domain, the TCR beta variable domain, and a TCR beta constant domain.

212. The method of claim 211, wherein the at least one tag is operably linked to the TCR beta variable domain.

213. The method of claim 212, wherein the at least one tag is an ALFA-tag.

214. The method of any one of claims 211-213, wherein the soluble TCR comprises a first linker.

215. The method of claim 214, wherein the at least one tag is operably linked to the TCR beta variable domain via the first linker.

216. The method of claim 214 or 215, wherein the first linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

217. The method of claim 216, wherein n is 1.

218. The method of any one of claims 211-217, wherein the soluble TCR comprises a second linker.

219. The method of claim 218, wherein the TCR alpha constant domain is operably linked to the at least one tag via the second linker.

220. The method of claim 218 or 219, wherein the second linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

221. The method of claim 216, wherein n is 4.

222. The method of any one of claims 207-221, wherein the soluble TCR comprises an additional tag operably linked to the C-terminus of the soluble TCR.

223. The method of claim 222, wherein the additional tag is a lOXHIS-sortag operably linked to the C-terminus of the soluble TCR.

224. The method of any one of claims 218-223, wherein the soluble TCR comprises, from N-terminus to C-terminus, the TCR alpha variable domain, the TCR alpha constant domain, the second linker, the at least one tag, the TCR beta variable region, the TCR beta constant region, and the additional tag.

225. The method of any one of claims 214-223, wherein the soluble TCR comprises, from N-terminus to C-terminus, the TCR alpha variable domain, the TCR alpha constant domain, the second linker, the at least one tag, the first linker, the TCR beta variable region, the TCR beta constant region, and the additional tag.

226. The method of any one of claims 196-225, wherein the soluble TCR comprises one or more disulfide bridges between the TCR alpha constant domain and the TCR beta constant domain.

227. The method of any one of claims 196-226, wherein the soluble TCR comprises a modification comprising removal of one or more N-glycosylation sites in the TCR alpha constant domain and / or TCR beta constant domain.

228. The method of any one of claims 196-227, wherein removal of the one or more N- glycosylation sites comprises substituting one or more asparagine residue to one or more aspartic acid residues or one or more glutamic acid residues.

229. The method of any one of claims 196-228, wherein the soluble TCR comprises a modification comprising removal of one or more unpaired cysteines in the TCR alpha constant domain and / or TCR beta constant domain.

230. The method of any one of claims 196-229, wherein removal of the one or more unpaired cysteines comprises substituting one or more unpaired cysteines to one or more serine residues.

231. The method of any one of claims 196-230, wherein the soluble TCR comprises one or more disulfide bonds in the TCR alpha constant domain and / or the TCR beta constant domain.

232. The method of any one of claims 196-231, wherein the soluble TCR comprises one or more mutations in the TCR alpha constant domain and / or the TCR beta constant domain.

233. The method of claim 232, wherein the one or more mutations are one or more stability enhancing mutations.

234. The method of claim 232 or 233, wherein the one or more mutations in the TCR alpha constant domain comprises S139F, T150I, or A190T.

235. The method of claim 232 or 233, wherein the one or more mutations in the TCR beta constant domain comprises C191A, N205D, E134K, H139R, D155P, or S170D.

236. The method of any one of claims 196-235, the TCR alpha constant domain and the TCR beta constant domain comprises one or more N>Q substitutions.

237. The method of any one of claims 196-231, wherein the soluble TCR comprises no mutations in the TCR alpha constant domain and / or the TCR beta constant domain.

238. The method of any one of claims 196-237, wherein the soluble TCR comprises a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 489.

239. The method of claim 238, wherein the soluble TCR comprises a sequence having a sequence set forth in SEQ ID NO: 489.

240. The method of claim 238 or 239, wherein the soluble TCR comprises a TCR alpha chain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 491.

241. The method of any one of claims 238-240, wherein the soluble TCR comprises a TCR alpha variable domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 421.

242. The method of any one of claims 238-241, wherein the soluble TCR comprises a TCR alpha constant domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 492.

243. The method of any one of claims 238-242, wherein the soluble TCR comprises a TCR beta chain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 132.

244. The method of any one of claims 238-243, wherein the soluble TCR comprises a TCR beta variable domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 427.

245. The method of any one of claims 238-244, wherein the soluble TCR comprises a TCR beta constant domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 490.

246. The method of any one of claims 211-237, wherein the soluble TCR comprises a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 494.

247. The method of claim 246, wherein the soluble TCR comprises a sequence having a sequence set forth in SEQ ID NO: 494.

248. The method of claim 246 or 247, wherein the soluble TCR comprises a TCR alpha chain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 496.

249. The method of any one of claims 246-248, wherein the soluble TCR comprises a TCR alpha variable domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 421.

250. The method of any one of claims 246-249, wherein the soluble TCR comprises a TCR alpha constant domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 492.

251. The method of any one of claims 246-250, wherein the soluble TCR comprises a TCR beta chain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 495.

252. The method of any one of claims 246-251, wherein the soluble TCR comprises a TCR beta variable domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 427.

253. The method of any one of claims 246-252, wherein the soluble TCR comprises a TCR beta constant domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 490.

254. The method of any one of claims 211-237, wherein the soluble TCR comprises a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 497.

255. The method of claim 254, wherein the soluble TCR comprises a sequence having a sequence set forth in SEQ ID NO: 497.

256. The method of claim 254 or 255, wherein the soluble TCR comprises a TCR alpha chain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 500.

257. The method of any one of claims 254-256, wherein the soluble TCR comprises a TCR alpha variable domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 421.

258. The method of any one of claims 254-257, wherein the soluble TCR comprises a TCR alpha constant domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 501.

259. The method of any one of claims 254-258, wherein the soluble TCR comprises a TCR beta chain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 498.

260. The method of any one of claims 254-259, wherein the soluble TCR comprises a TCR beta variable domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 427.

261. The method of any one of claims 254-260, wherein the soluble TCR comprises a TCR beta constant domain comprising a sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 499.

262. The method of any one of claims 196-237, wherein the TCR alpha variable domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR alpha variable domain sequences of Tables 5, 6, 8, 11, and 12.

263. The method of claim 262, wherein the TCR beta variable domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR beta variable domain sequences of Tables 5, 6, 8, 11, and 12.

264. The method of claim 238 or 263, wherein the TCR alpha constant domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR alpha constant domain sequences of Tables 5, 6, 8, 11, and 12.

265. The method of claim 264, wherein the TCR beta constant domain comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of the TCR beta constant domain sequences of Tables 5, 6, 8, 11, 11, and 12.

266. The method of any one of claims 222-265, wherein the additional tag comprises a sequence of SEQ ID NO : 131.

267. The method of any one of claims 175-266, wherein the at least one tag comprises a sequence of SEQ ID NO: 126, or a sequence that comprises less than 3 amino acids differing from the above within the sequence.

268. The method of any one of claims 175-267, wherein the soluble TCR exhibits anti-tumor cytotoxicity at a level comparable to a corresponding heterodimeric soluble TCR.

269. The method of any one of claims 175-267, wherein the soluble TCR induces T cell proliferation at a level comparable to a corresponding heterodimeric soluble TCR.

270. The method of any one of claims 175-267, wherein the soluble TCR upregulates expression of one or more cytotoxicity markers at a level comparable to a corresponding heterodimeric soluble TCR.

271. The method of any one of claims 175-270, wherein the soluble TCR binds to an epitope on the surface of the target cell.

272. The method of claim 271, wherein the epitope is derived from a RAS polypeptide or a fragment thereof.

273. The method of claim 272, wherein the RAS polypeptide is a mutated RAS polypeptide.

274. The method of claim 271, wherein the epitope is derived from a GAT A3 polypeptide or a fragment thereof.

275. The method of any one of claims 191-274, wherein the single chain TCR comprises a linker between the TCR alpha variable domain and the TCR beta variable domain.

276. The method of claim 275, wherein the linker is a polypeptide linker.

277. The method of claim 275 or 276, wherein the linker comprises the at least one tag sequence.

278. The method of any one of claims 275-277, wherein the linker comprises a sequence selected from the group consisting of SEQ ID NOs: 125, 128, 135, and 488.

279. The method of any one of claims 191-278, wherein the soluble TCR is a single chain TCR, and wherein the single chain TCR comprises, from N terminus to C terminus, the TCR alpha variable domain, the TCR alpha constant domain, a first linker, a first tag, a second linker, a second tag, the TCR beta variable domain, and the TCR beta constant domain.

280. The method of claim 279, wherein the first tag or the second tag comprises a sequence of SEQ ID NO: 126.

281. The method of claim 279 or 280, wherein the first tag and the second tag each comprises a sequence of SEQ ID NO: 126.

282. The method of any one of claims 279-281, wherein the first linker or the second linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

283. The method of claim 282, wherein n is 2 or 4.

284. The method of any one of claims 279-283, wherein the first linker comprises a sequence of (GGGGS)4, and the second linker comprises a sequence of GGGGS.

285. The method of any one of claims 279-283, wherein the first linker comprises a sequence of (GGGGS)2, and the second linker comprises a sequence of GGGGS.

286. The method of any one of claims 191-285, wherein the soluble TCR comprises a sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 552, 556, and 557.

287. The method of any one of claims 187-190 and 271-274, wherein the TCR alpha variable domain and the TCR beta variable domain are on two separate polypeptide chains comprising a first polypeptide chain and a second polypeptide chain.

288. The method of claim 287, wherein the first polypeptide chain and / or the second polypeptide chain comprises a sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 138, 541, 543, 544, 545, 547, 548, and 550.

289. The method of claim 287 or 288, wherein the first polypeptide chain comprises a first dimerizing domain fused to the TCR alpha variable domain, and the second polypeptide chain comprises a second dimerizing domain fused to the TCR beta variable domain.

290. The method of claim 289, wherein the first dimerizing domain or the second dimerizing domain comprises at least 2, at least 5, at least 10 or more amino acids of a TCR extracellular domain adjacent to a TCR transmembrane domain.

291. The method of claim 289 or 290, wherein the first dimerizing domain comprises a sequence as set forth in SEQ ID NO 425.

292. The method of claim 289 or 290, wherein the second dimerizing domain comprises a sequence as set forth in SEQ ID NO: 431.

293. The method of any one of claims 287-291, wherein the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain and the first dimerizing domain; and the second polypeptide comprises, from N terminus to C terminus, the TCR beta variable domain and the second dimerizing domain.

294. The method of any one of claims 287-293, wherein the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, a first peptide linker, and the first dimerizing domain; and the second polypeptide comprises, from N terminus to C terminus, the TCR beta variable domain, a second peptide linker, and the second dimerizing domain.

295. The method of any one of claims 287-291, wherein the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain and the TCR alpha variable domain, and the second polypeptide comprises, from N terminus to C terminus, the second dimerizing domain and the TCR beta variable domain.

296. The method of any one of claims 287-291 and 295, wherein the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain, a firstpeptide linker, and the TCR alpha variable domain, and the second polypeptide comprises, from N terminus to C terminus, the second dimerizing domain, a second peptide linker, and the TCR beta variable domain.

297. The method of any one of claims 289-296, wherein the first dimerizing domain comprises a TCR alpha constant (TRAC) domain or portion thereof; and the second dimerizing domain comprises a TCR beta constant (TRBC) domain or portion thereof.

298. The method of any one of claims 289-297, wherein the first dimerizing domain and the second dimerizing domain are linked by one or more disulfide bridges.

299. The method of claim 298, wherein the first dimerizing domain and the second dimerizing domain are linked by a single disulfide bridge.

300. The method of any one of claims 287-299, wherein the first polypeptide chain comprises the at least one tag.

301. The method of claim 300, wherein the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, and the at least one tag.

302. The method of claim 301, wherein the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, a linker, and the at least one tag.

303. The method of claim 300, wherein the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the TCR alpha variable domain, and the first dimerizing domain.

304. The method of claim 303, wherein the first polypeptide chain comprises, from N terminus to C terminus, the at least one tag, a linker, the TCR alpha variable domain, and the first dimerizing domain.

305. The method of claim 302 or 304, wherein the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

306. The method of claim 300, wherein the first polypeptide chain comprises, from N terminus to C terminus, the first dimerizing domain, the TCR alpha variable domain, and the at least one tag.

307. The method of claim 300, wherein the first polypeptide chain comprises, from N terminus to C terminus, the tag, the first dimerizing domain, and the TCR alpha variable domain.

308. The method of any one of claims 287-299, wherein the second polypeptide chain comprises the at least one tag.

309. The method of claim 308, wherein the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, the TCR beta variable domain, and the second dimerizing domain.

310. The method of claim 309, wherein the second polypeptide chain comprises, from N terminus to C terminus, the at least one tag, a linker, the TCR beta variable domain, and the second dimerizing domain.

311. The method of claim 308, wherein the second polypeptide comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, and the at least one tag.

312. The method of claim 311, wherein the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, a linker, and the at least one tag.

313. The method of claim 310 or 312, wherein the linker comprises a sequence of (GGGGS)n, wherein n is an integer from 1 to 10.

314. The method of claim 308, wherein the second polypeptide comprises, from N terminus to C terminus, the second dimerizing domain, the TCR alpha variable domain, and the at least one tag.

315. The method of claim 308, wherein the second polypeptide comprises, from N terminus to C terminus, the at least one tag, the second dimerizing domain, and the TCR beta variable domain.

316. The method of any one of claims 175-315, wherein the at least one tag comprises two or more tags.

317. The method of claim 316, wherein the at least one tag comprises two tags.

318. The method of claim 316, wherein the at least one tag comprises three tags.

319. The method of any one of claims 316-318, wherein each tag of the two or more tags comprises a sequence of SEQ ID NO: 126.

320. The method of any one of claims 316-319, wherein the two or more tags are operably linked by a linker having a sequence of (GGGGS)n, where n is an integer from 1 to 10.

321. The method of any one of claims 316-320, wherein the two or more tags are linked to the same first polypeptide chain or the same second polypeptide chain.

322. The method of any one of claims 316-320, wherein the two or more tags are linked to different polypeptide chains.

323. The method of any one of claims 318-322, wherein the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, and the two or more tags operably linked.

324. The method of claim 323, wherein the first polypeptide chain comprises, from N terminus to C terminus, the TCR alpha variable domain, the first dimerizing domain, a first linker, a first tag, a second linker, and a second tag.

325. The method of any one of claims 318-322, wherein the first polypeptide chain comprises, from N terminus to C terminus, the two or more tags operably linked, the TCR alpha variable domain, and the first dimerizing domain.

326. The method of claim 325, wherein the first polypeptide chain comprises, from N terminus to C terminus, a first tag, a first linker, a second tag, a second linker, the TCR alpha variable domain, and the first dimerizing domain.

327. The method of any one of claims 318-322, wherein the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, and the two or more tags operably linked.

328. The method of claim 327, wherein the second polypeptide chain comprises, from N terminus to C terminus, the TCR beta variable domain, the second dimerizing domain, a first linker, a first tag, a second linker, and a second tag.

329. The method of any one of claims 318-322, wherein the second polypeptide chain comprises, from N terminus to C terminus, the two or more tags operably linked, the TCR beta variable domain, and the second dimerizing domain.

330. The method of claim 329, wherein the second polypeptide chain comprises, from N terminus to C terminus, a first tag, a first linker, a second tag, a second linker, the TCR beta variable domain, and the second dimerizing domain.

331. The method of any one of claims 324, 326, 328 and 330, wherein the first tag and the second tag comprise the same sequence.

332. The method of claim 331, wherein the sequence comprises a sequence of SEQ ID NO: 126.

333. The method of any one of claims 324, 326, 328 and 330-332, wherein the first linker or the second linker comprises a sequence of (GGGGS)n, where n is an integer from 1 to 10.

334. The method of any one of claims 175-333, wherein the soluble TCR comprises two or more soluble TCRs, wherein each soluble TCR of the two or more soluble TCRs recognizes a different peptide:MHC complex, wherein (i) different peptide:MHC complexes are on the same target cell or different target cells, (ii) different peptide:MHC complexes comprise different epitope sequences, and / or (iii) different peptide:MHC complexes comprise different MHC molecules encoded by different HLA alleles.

335. The method of claim 334, wherein the different peptide:MHC complexes comprise different epitope sequences presented by the same MHC molecule encoded by the same HLA allele.

336. The method of claim 334 or 335, wherein the different peptideMHC complexes comprise different epitope sequences presented by different MHC molecules encoded by different HLA alleles.

337. The method of any one of claims 334-336, wherein the different peptide:MHC complexes comprise the same epitope sequence presented by different MHC molecule encoded by different HLA alleles.

338. The method of any one of claims 334-337, wherein the different peptide:MHC complexes are on the same target cell.

339. The method of any one of claims 334-338, wherein the different peptide:MHC complexes are on different target cells.

Images