T cell receptors and methods of use thereof

Abstract

The present disclosure relates to recombinant T cell receptors capable of binding to a tyrosinase epitope, a MAGA-A1 epitope, a MART1 epitope, a MAGE-A3 epitope, or a SSX2 epitope, and nucleic acid molecules encoding the same. In some embodiments, the nucleic acid molecule further comprises a second nucleotide sequence, wherein the second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits the expression of an endogenous TCR. Other aspects of the present disclosure relate to vectors comprising the nucleic acid molecules and cells comprising the recombinant TCRs, the nucleic acid molecules, or the vectors. Other aspects of the present disclosure relate to methods of their use. In some embodiments, the methods comprise treating a cancer in a subject in need thereof.

Description

[0001] Cross-references to related applications

[0002] This PCT application claims priority to U.S. Provisional Patent Application No. 62 / 823,487, filed March 25, 2019, which is incorporated herein by reference in its entirety.

[0003] References to sequence listings submitted electronically via EFS-WEB

[0004] The contents of the sequence list submitted electronically (name: 4285_008PC01_SeqListing_ST25.txt, size: 69,638 bytes; and creation date: March 23, 2020) are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure provides a recombinant T-cell receptor (“TCR”) that specifically binds to target human proteins selected from the group consisting of tyrosinase, MAGE-A1, MART1, MAGE-A3, and SSX2, and its uses therein. Background Technology

[0006] Immunotherapy has become an important tool in the fight against a variety of diseases, including cancer. T-cell therapy is at the forefront of immunotherapy development and has been shown to induce clinical responses in cancer patients through adoptive transfer of anti-tumor T cells. Although many T-cell therapies target mutated tumor antigens, the vast majority of these neoantigens are not common and are unique to each patient.

[0007] The number of potential non-mutated antigens is orders of magnitude higher than that of mutated antigens. Elucidating T-cell epitopes derived from shared antigens can facilitate the robust development of effective and safe adoptive T-cell therapies that are readily available to a large population of cancer patients. However, the sheer number of non-mutated antigens and the high degree of HLA gene polymorphism can hinder comprehensive analysis of the specificity of anti-tumor T-cell responses to non-mutated antigens.

[0008] This disclosure provides novel epitopes of nonmutated antigen tyrosinases, MAGE-A1, MART1, MAGE-A3, and SSX2, and TCRs capable of specifically binding to said epitopes. These novel epitopes are associated with specific HLA alleles. Using these tumor-responsive HLA-restricted TCRs helps expand the applicability of TCR gene therapy, particularly in immuno-oncology. Summary of the Invention

[0009] Certain aspects of the present disclosure relate to a nucleic acid molecule comprising (i) a first nucleotide sequence encoding a recombinant T cell receptor (TCR) or an antigen binding portion thereof that specifically binds to a target human protein; and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits expression of an endogenous TCR; wherein: (a) the target human protein is tyrosinase (“anti-tyrosinase TCR”), wherein the anti-tyrosinase TCR cross-competes for binding to human tyrosinase with a reference anti-tyrosinase TCR, wherein the reference anti-tyrosinase TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 1, and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 2; (b) the target human protein is MAGE-A1 (“anti-MAGE-A1 TCR”), wherein the anti-MAGE-A1 TCR cross-competes for binding to human MAGE-A1 with a reference anti-MAGE-A1 TCR, wherein the reference anti-MAGE-A1 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 11, and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 12; (c) the target human protein is MART1 (“anti-MART1 TCR”), wherein the anti-MART1 TCR cross-competes for binding to human MART1 with a reference anti-MART1 TCR, wherein the reference anti-MART1 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 21, and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 22; (d) the target human protein is MAGE-A3 (“anti-MAGE-A3 TCR”), wherein the anti-MAGE-A3 TCR cross-competes for binding to human MAGE-A3 with a reference anti-MAGE-A3 TCR, wherein the reference anti-MAGE-A3 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 31, and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 32; or (e) the target human protein is SSX2 (“anti-SSX2 TCR”), wherein the anti-SSX2 TCR cross-competes for binding to human SSX2 with a reference anti-SSX2 TCR, wherein the reference anti-SSX2 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 41, and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 42.

[0010] Certain aspects of the present disclosure relate to a nucleic acid molecule comprising (i) a first nucleotide sequence encoding a recombinant T cell receptor (TCR) or an antigen binding portion thereof that specifically binds to a target human protein; and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits expression of an endogenous TCR; wherein: (a) the target human protein is tyrosinase (“anti-tyrosinase TCR”), wherein the anti-tyrosinase TCR binds to the same epitope or an overlapping epitope of human tyrosinase as a reference anti-tyrosinase TCR, wherein the reference anti-tyrosinase TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 2; (b) the target human protein is MAGE-A1 (“anti-MAGE-A1 TCR”), wherein the anti-MAGE-A1 TCR binds to the same epitope or an overlapping epitope of human MAGE-A1 as a reference anti-MAGE-A1 TCR, wherein the reference anti-MAGE-A1 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 12; (c) the target human protein is MART1 (“anti-MART1 TCR”), wherein the anti-MART1 TCR binds to the same epitope or an overlapping epitope of human MART1 as a reference anti-MART1 TCR, wherein the reference anti-MART1 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 21 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 22; (d) the target human protein is MAGE-A3 (“anti-MAGE-A3 TCR”), wherein the anti-MAGE-A3 TCR binds to the same epitope or an overlapping epitope of human MAGE-A3 as a reference anti-MAGE-A3 TCR, wherein the reference anti-MAGE-A3 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 31 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 32; or (e) the target human protein is SSX2 (“anti-SSX2 TCR”), wherein the anti-SSX2 TCR binds to the same epitope or an overlapping epitope of human SSX2 as a reference anti-SSX2 TCR, wherein the reference anti-SSX2 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 41 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 42.

[0011] In some embodiments, (a) the anti-tyrosinase TCR binds to an epitope of tyrosinase, the epitope consisting of the amino acid sequence as set forth in SEQ ID NO: 51; (b) the anti-MAGE-A1 TCR binds to an epitope of MAGE-A1, the epitope consisting of the amino acid sequence as set forth in SEQ ID NO: 52; (c) the anti-MART1 TCR binds to an epitope of MART1, the epitope consisting of the amino acid sequence as set forth in SEQ ID NO: 53; (d) the anti-MAGE-A3 TCR binds to an epitope of MAGE-A3, the epitope consisting of the amino acid sequence as set forth in SEQ ID NO: 54; or (e) the anti-SSX2 TCR binds to an epitope of SSX2, the epitope consisting of the amino acid sequence as set forth in SEQ ID NO: 55.

[0012] In some embodiments, the epitope is complexed with an HLA class I molecule. In some embodiments, the HLA class I molecule is an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G allele. In some embodiments, (a) the target human protein is tyrosinase and the HLA class I molecule is an HLA-C*05 allele; (b) the target human protein is MAGE-A1 and the HLA class I molecule is an HLA-B*07 allele; (c) the target human protein is MART1 and the HLA class I molecule is an HLA-B*18 allele; (d) the target human protein is MAGE-A3 and the HLA class I molecule is an HLA-B*18 allele; or (e) the target human protein is SSX2 and the HLA class I molecule is an HLA-A*02 allele.

[0013] In some embodiments, (a) the target human protein is tyrosinase and the HLA Class I molecule is selected from the group consisting of an HLA-C*05:01 allele, an HLA-C*05:03 allele, an HLA-C*05:04 allele, an HLA-C*05:05 allele, and an HLA-C*05:06 allele; (b) the target human protein is MAGE-A1 and the HLA Class I molecule is selected from the group consisting of an HLA-B*07:02 allele, an HLA-B*07:03 allele, an HLA-B*07:04 allele, an HLA-B*07:05 allele, and an HLA-B*07:06 allele; (c) the target human protein is MART1 and the HLA Class I molecule is selected from the group consisting of an HLA-B*18:01 allele, an HLA-B*18:02 allele, an HLA-B*18:03 allele, an HLA-B*18:04 allele, and an HLA-B*18:05 allele; (d) the target human protein is MAGE-A3 and the HLA Class I molecule is selected from the group consisting of an HLA-B*18:01 allele, an HLA-B*18:02 allele, an HLA-B*18:03 allele, an HLA-B*18:04 allele, and an HLA-B*18:05 allele; or (e) the target human protein is SSX2 and the HLA Class I molecule is selected from the group consisting of an HLA-A*02:01 allele, an HLA-A*02:02 allele, an HLA-A*02:03 allele, an HLA-A*02:04 allele, and an HLA-A*02:05 allele.

[0014] In some embodiments, (a) the target human protein is tyrosinase and the HLA Class I molecule is an HLA-C*05:01 allele; (b) the target human protein is MAGE-A1 and the HLA Class I molecule is an HLA-B*07:02 allele; (c) the target human protein is MART1 and the HLA Class I molecule is an HLA-B*18:01 allele; (d) the target human protein is MAGE-A3 and the HLA Class I molecule is an HLA-B*18:01 allele; or (e) the target human protein is SSX2 and the HLA Class I molecule is an HLA-A*02:01 allele.

[0015] In some embodiments, the recombinant TCR or antigen binding portion thereof that specifically binds to a target human protein comprises an alpha chain and a beta chain; wherein the alpha chain comprises a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3; wherein the beta chain comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; and wherein: (a) the target human protein is tyrosinase, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 7; (b) the target human protein is MAGE-A1, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 17; (c) the target human protein is MART1, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 27; (d) the target human protein is MAGE-A3, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 37; or (e) the target human protein is SSX2, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 47.

[0016] In some embodiments, (a) the target human protein is tyrosinase, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 10; (b) the target human protein is MAGE-A1, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 20; (c) the target human protein is MART1, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 30; (d) the target human protein is MAGE-A3, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 40; or (e) the target human protein is SSX2, and the alpha beta CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 50.

[0017] In some embodiments, the recombinant TCR or antigen binding portion thereof that specifically binds to a target human protein comprises an alpha chain and a beta chain, wherein the alpha chain comprises a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3; wherein the beta chain comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; and wherein: (a) the target human protein is tyrosinase, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 10; (b) the target human protein is MAGE-A1, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 20; (c) the target human protein is MART1, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 30; (d) the target human protein is MAGE-A3, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 40; or (e) the target human protein is SSX2, and the alpha beta CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 50.

[0018] In some embodiments, (a) the target human protein is tyrosinase, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 7; (b) the target human protein is MAGE-A1, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 17; (c) the target human protein is MART1, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 27; (d) the target human protein is MAGE-A3, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 37; or (e) the target human protein is SSX2, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 47.

[0019] In some embodiments, (a) the target human protein is tyrosinase, and the alpha chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 5; (b) the target human protein is MAGE-A1, and the alpha chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 15; (c) the target human protein is MART1, and the alpha chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 25; (d) the target human protein is MAGE-A3, and the alpha chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 35; or (e) the target human protein is SSX2, and the alpha chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 45.

[0020] In some embodiments, (a) the target human protein is tyrosinase, and the beta chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 8; (b) the target human protein is MAGE-A1, and the beta chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 18; (c) the target human protein is MART1, and the beta chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 28; (d) the target human protein is MAGE-A3, and the beta chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 38; or (e) the target human protein is SSX2, and the beta chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 48.

[0021] In some embodiments, (a) the target human protein is tyrosinase, and the alpha chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 6; (b) the target human protein is MAGE-A1, and the alpha chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 16; (c) the target human protein is MART1, and the alpha chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 26; (d) the target human protein is MAGE-A3, and the alpha chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 36; or (e) the target human protein is SSX2, and the alpha chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 46.

[0022] In some embodiments, (a) the target human protein is tyrosinase, and the beta chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 9; (b) the target human protein is MAGE-A1, and the beta chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 19; (c) the target human protein is MART1, and the beta chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 29; (d) the target human protein is MAGE-A3, and the beta chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 39; or (e) the target human protein is SSX2, and the beta chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 49.

[0023] In some embodiments, (a) the target human protein is tyrosinase and the alpha chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 1; (b) the target human protein is MAGE-A1 and the alpha chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 11; (c) the target human protein is MART1 and the alpha chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 21; (d) the target human protein is MAGE-A3 and the alpha chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 31; or (e) the target human protein is SSX2 and the alpha chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 41.

[0024] In some embodiments, (a) the target human protein is tyrosinase and the beta chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 2; (b) the target human protein is MAGE-A1 and the beta chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 12; (c) the target human protein is MART1 and the beta chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 22; (d) the target human protein is MAGE-A3 and the beta chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 32; or (e) the target human protein is SSX2 and the beta chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the alpha chain further comprises a constant region, wherein the constant region is different from the endogenous constant region of the alpha chain.

[0025] In some embodiments, the alpha chain further comprises a constant region, wherein: (a) the target human protein is tyrosinase, and the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 1; (b) the target human protein is MAGE-A1, and the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 11; (c) the target human protein is MART1, and the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 21; (d) the target human protein is MAGE-A3, and the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 31; or (e) the target human protein is SSX2, and the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 41.

[0026] In some embodiments, (a) the target human protein is tyrosinase and the alpha chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO: 1; (b) the target human protein is MAGE-A1 and the alpha chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO: 11; (c) the target human protein is MART1 and the alpha chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO: 21; (d) the target human protein is MAGE-A3 and the alpha chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO: 31; or (e) the target human protein is SSX2 and the alpha chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the beta chain further comprises a constant region, wherein the constant region is different from the endogenous constant region of the beta chain.

[0027] In some embodiments, the beta chain further comprises a constant region, wherein: (a) the target human protein is tyrosinase, and the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 2; (b) the target human protein is MAGE-A1, and the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 12; (c) the target human protein is MART1, and the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 22; (d) the target human protein is MAGE-A3, and the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 32; or (e) the target human protein is SSX2, and the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 42.

[0028] In some embodiments, (a) the target human protein is tyrosinase, and the beta chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to a constant region present in the amino acid sequence set forth in SEQ ID NO: 2; (b) the target human protein is MAGE-A1, and the beta chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to a constant region present in the amino acid sequence set forth in SEQ ID NO: 12; (c) the target human protein is MART1, and the beta chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to a constant region present in the amino acid sequence set forth in SEQ ID NO: 22; (d) the target human protein is MAGE-A3, and the beta chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to a constant region present in the amino acid sequence set forth in SEQ ID NO: 32; or (e) the target human protein is SSX2, and the beta chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to a constant region present in the amino acid sequence set forth in SEQ ID NO: 42.

[0029] In some embodiments, (a) the target human protein is tyrosinase, and the alpha chain comprises the amino acid sequence as set forth in SEQ ID NO: 1; (b) the target human protein is MAGE-A1, and the alpha chain comprises the amino acid sequence as set forth in SEQ ID NO: 11; (c) the target human protein is MART1, and the alpha chain comprises the amino acid sequence as set forth in SEQ ID NO: 21; (d) the target human protein is MAGE-A3, and the alpha chain comprises the amino acid sequence as set forth in SEQ ID NO: 31; or (e) the target human protein is SSX2, and the alpha chain comprises the amino acid sequence as set forth in SEQ ID NO: 41.

[0030] In some embodiments, (a) the target human protein is tyrosinase, and the beta chain comprises the amino acid sequence as set forth in SEQ ID NO: 2; (b) the target human protein is MAGE-A1, and the beta chain comprises the amino acid sequence as set forth in SEQ ID NO: 12; (c) the target human protein is MART1, and the beta chain comprises the amino acid sequence as set forth in SEQ ID NO: 22; (d) the target human protein is MAGE-A3, and the beta chain comprises the amino acid sequence as set forth in SEQ ID NO: 32; or (e) the target human protein is SSX2, and the beta chain comprises the amino acid sequence as set forth in SEQ ID NO: 42.

[0031] In some embodiments, the second nucleotide sequence is one or more siRNAs that reduce expression of an endogenous TCR. In some embodiments, the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of an endogenous TCR. In some embodiments, the one or more siRNAs comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 57-60. In some embodiments, the second nucleotide sequence encodes a Cas9.

[0032] In some embodiments, the recombinant TCR or antigen-binding portion thereof comprises an alpha chain constant region, a beta chain constant region, or both; and wherein the alpha chain constant region, the beta chain constant region, or both comprise an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 substitutions within a target sequence relative to a corresponding amino acid sequence of an endogenous TCR.

[0033] Certain aspects of the present disclosure relate to a vector comprising a nucleic acid molecule disclosed herein. In some embodiments, the vector is a viral vector, a mammalian vector, or a bacterial vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is selected from the group consisting of an adenoviral vector, a lentivirus, a Sendai virus vector, a baculovirus vector, an Epstein Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, a hybrid vector, and an adeno-associated virus (AAV) vector. In some embodiments, the vector is a lentivirus.

[0034] Certain aspects of the present disclosure relate to a T cell receptor (TCR) or antigen-binding portion thereof comprising an alpha chain variable domain of a recombinant TCR or antigen-binding portion thereof disclosed herein and a beta chain variable domain of a recombinant TCR or antigen-binding portion thereof disclosed herein.

[0035] Certain aspects of the present disclosure relate to a recombinant T cell receptor (TCR) or an antigen binding portion thereof that specifically binds to a target human protein ("recombinant TCR"), wherein: (a) the target human protein is tyrosinase ("anti-tyrosinase TCR"), wherein the anti-tyrosinase TCR cross-competes for binding to human tyrosinase with a reference anti-tyrosinase TCR, wherein the reference anti-tyrosinase TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 2; (b) the target human protein is MAGE-A1 ("anti-MAGE-A1 TCR"), wherein the anti-MAGE-A1 TCR cross-competes for binding to human MAGE-A1 with a reference anti-MAGE-A1 TCR, wherein the reference anti-MAGE-A1 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 12; (c) the target human protein is MART1 ("anti-MART1 TCR"), wherein the anti-MART1 TCR cross-competes for binding to human MART1 with a reference anti-MART1 TCR, wherein the reference anti-MART1 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 21 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 22; (d) the target human protein is MAGE-A3 ("anti-MAGE-A3 TCR"), wherein the anti-MAGE-A3 TCR cross-competes for binding to human MAGE-A3 with a reference anti-MAGE-A3 TCR, wherein the reference anti-MAGE-A3 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 31 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 32; or (e) the target human protein is SSX2 ("anti-SSX2 TCR"), wherein the anti-SSX2 TCR cross-competes for binding to human SSX2 with a reference anti-SSX2 TCR, wherein the reference anti-SSX2 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 41 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 42.

[0036] Certain aspects of the present disclosure relate to a recombinant T cell receptor (TCR) or an antigen binding portion thereof that specifically binds to a target human protein ("recombinant TCR"), wherein: (a) the target human protein is tyrosinase ("anti-tyrosinase TCR"), wherein the anti-tyrosinase TCR binds to the same epitope or an overlapping epitope of human tyrosinase as a reference anti-tyrosinase TCR, wherein the reference anti-tyrosinase TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 2; (b) the target human protein is MAGE-A1 ("anti-MAGE-A1 TCR"), wherein the anti-MAGE-A1 TCR binds to the same epitope or an overlapping epitope of human MAGE-A1 as a reference anti-MAGE-A1 TCR, wherein the reference anti-MAGE-A1 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 12; (c) the target human protein is MART1 ("anti-MART1 TCR"), wherein the anti-MART1 TCR binds to the same epitope or an overlapping epitope of human MART1 as a reference anti-MART1 TCR, wherein the reference anti-MART1 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 21 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 22; (d) the target human protein is MAGE-A3 ("anti-MAGE-A3 TCR"), wherein the anti-MAGE-A3 TCR binds to the same epitope or an overlapping epitope of human MAGE-A3 as a reference anti-MAGE-A3 TCR, wherein the reference anti-MAGE-A3 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 31 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 32; or (e) the target human protein is SSX2 ("anti-SSX2 TCR"), wherein the anti-SSX2 TCR binds to the same epitope or an overlapping epitope of human SSX2 as a reference anti-SSX2 TCR, wherein the reference anti-SSX2 TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 41 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 42.

[0037] In some embodiments, (a) the anti-tyrosinase TCR binds to an epitope of tyrosinase, the epitope consisting of the amino acid sequence as set forth in SEQ ID NO: 51; (b) the anti-MAGE-A1 TCR binds to an epitope of MAGE-A1, the epitope consisting of the amino acid sequence as set forth in SEQ ID NO: 52; (c) the anti-MART1 TCR binds to an epitope of MART1, the epitope consisting of the amino acid sequence as set forth in SEQ ID NO: 53; (d) the anti-MAGE-A3 TCR binds to an epitope of MAGE-A3, the epitope consisting of the amino acid sequence as set forth in SEQ ID NO: 54; or (e) the anti-SSX2 TCR binds to an epitope of SSX2, the epitope consisting of the amino acid sequence as set forth in SEQ ID NO: 55.

[0038] In some embodiments, the epitope is complexed with an HLA class I molecule. In some embodiments, the HLA class I molecule is an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G allele. In some embodiments: (a) the target human protein is tyrosinase, and the HLA class I molecule is an HLA-C*05 allele; (b) the target human protein is MAGE-A1, and the HLA class I molecule is an HLA-B*07 allele; (c) the target human protein is MART1, and the HLA class I molecule is an HLA-B*18 allele; (d) the target human protein is MAGE-A3, and the HLA class I molecule is an HLA-B*18 allele; or (e) the target human protein is SSX2, and the HLA class I molecule is an HLA-A*02 allele.

[0039] In some embodiments, (a) the target human protein is tyrosinase and the HLA Class I molecule is selected from the group consisting of an HLA-C*05:01 allele, an HLA-C*05:03 allele, an HLA-C*05:04 allele, an HLA-C*05:05 allele, and an HLA-C*05:06 allele; (b) the target human protein is MAGE-A1 and the HLA Class I molecule is selected from the group consisting of an HLA-B*07:02 allele, an HLA-B*07:03 allele, an HLA-B*07:04 allele, an HLA-B*07:05 allele, and an HLA-B*07:06 allele; (c) the target human protein is MART1 and the HLA Class I molecule is selected from the group consisting of an HLA-B*18:01 allele, an HLA-B*18:02 allele, an HLA-B*18:03 allele, an HLA-B*18:04 allele, and an HLA-B*18:05 allele; (d) the target human protein is MAGE-A3 and the HLA Class I molecule is selected from the group consisting of an HLA-B*18:01 allele, an HLA-B*18:02 allele, an HLA-B*18:03 allele, an HLA-B*18:04 allele, and an HLA-B*18:05 allele; or (e) the target human protein is SSX2 and the HLA Class I molecule is selected from the group consisting of an HLA-A*02:01 allele, an HLA-A*02:02 allele, an HLA-A*02:03 allele, an HLA-A*02:04 allele, and an HLA-A*02:05 allele.

[0040] In some embodiments, (a) the target human protein is tyrosinase and the HLA Class I molecule is an HLA-C*05:01 allele; (b) the target human protein is MAGE-A1 and the HLA Class I molecule is an HLA-B*07:02 allele; (c) the target human protein is MART1 and the HLA Class I molecule is an HLA-B*18:01 allele; (d) the target human protein is MAGE-A3 and the HLA Class I molecule is an HLA-B*18:01 allele; or (e) the target human protein is SSX2 and the HLA Class I molecule is an HLA-A*02:01 allele.

[0041] In some embodiments, the recombinant TCR or antigen binding portion thereof that specifically binds to a target human protein comprises an alpha chain and a beta chain; wherein the alpha chain comprises a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3; wherein the beta chain comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; and wherein: (a) the target human protein is tyrosinase, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 7; (b) the target human protein is MAGE-A1, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 17; (c) the target human protein is MART1, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 27; (d) the target human protein is MAGE-A3, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 37; or (e) the target human protein is SSX2, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 47.

[0042] In some embodiments, (a) the target human protein is tyrosinase, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 10; (b) the target human protein is MAGE-A1, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 20; (c) the target human protein is MART1, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 30; (d) the target human protein is MAGE-A3, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 40; or (e) the target human protein is SSX2, and the alpha beta CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 50.

[0043] In some embodiments, the recombinant TCR or antigen binding portion thereof that specifically binds to a target human protein comprises an alpha chain and a beta chain, wherein the alpha chain comprises a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3; wherein the beta chain comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; and wherein: (a) the target human protein is tyrosinase, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 10; (b) the target human protein is MAGE-A1, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 20; (c) the target human protein is MART1, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 30; (d) the target human protein is MAGE-A3, and the beta chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 40; or (e) the target human protein is SSX2, and the alpha beta CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 50.

[0044] In some embodiments, (a) the target human protein is tyrosinase, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 7; (b) the target human protein is MAGE-A1, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 17; (c) the target human protein is MART1, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 27; (d) the target human protein is MAGE-A3, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 37; or (e) the target human protein is SSX2, and the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 47.

[0045] In some embodiments, (a) the target human protein is tyrosinase, and the alpha chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 5; (b) the target human protein is MAGE-A1, and the alpha chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 15; (c) the target human protein is MART1, and the alpha chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 25; (d) the target human protein is MAGE-A3, and the alpha chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 35; or (e) the target human protein is SSX2, and the alpha chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 45.

[0046] In some embodiments, (a) the target human protein is tyrosinase, and the beta chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 8; (b) the target human protein is MAGE-A1, and the beta chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 18; (c) the target human protein is MART1, and the beta chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 28; (d) the target human protein is MAGE-A3, and the beta chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 38; or (e) the target human protein is SSX2, and the beta chain CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 48.

[0047] In some embodiments, (a) the target human protein is tyrosinase, and the alpha chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 6; (b) the target human protein is MAGE-A1, and the alpha chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 16; (c) the target human protein is MART1, and the alpha chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 26; (d) the target human protein is MAGE-A3, and the alpha chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 36; or (e) the target human protein is SSX2, and the alpha chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 46.

[0048] In some embodiments, (a) the target human protein is tyrosinase, and the beta chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 9; (b) the target human protein is MAGE-A1, and the beta chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 19; (c) the target human protein is MART1, and the beta chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 29; (d) the target human protein is MAGE-A3, and the beta chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 39; or (e) the target human protein is SSX2, and the beta chain CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 49.

[0049] In some embodiments, (a) the target human protein is tyrosinase and the alpha chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 1; (b) the target human protein is MAGE-A1 and the alpha chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 11; (c) the target human protein is MART1 and the alpha chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 21; (d) the target human protein is MAGE-A3 and the alpha chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 31; or (e) the target human protein is SSX2 and the alpha chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 41.

[0050] In some embodiments, (a) the target human protein is tyrosinase and the beta chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 2; (b) the target human protein is MAGE-A1 and the beta chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 12; (c) the target human protein is MART1 and the beta chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 22; (d) the target human protein is MAGE-A3 and the beta chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 32; or (e) the target human protein is SSX2 and the beta chain variable domain comprises the amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 42.

[0051] In some embodiments, the alpha chain further comprises a constant region, wherein: (a) the target human protein is tyrosinase, and the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 1; (b) the target human protein is MAGE-A1, and the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 11; (c) the target human protein is MART1, and the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 21; (d) the target human protein is MAGE-A3, and the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 31; or (e) the target human protein is SSX2, and the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 41.

[0052] In some embodiments, the beta chain further comprises a constant region, and wherein: (a) the target human protein is tyrosinase, and the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 2; (b) the target human protein is MAGE-A1, and the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 12; (c) the target human protein is MART1, and the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 22; (d) the target human protein is MAGE-A3, and the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 32; or (e) the target human protein is SSX2, and the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 42.

[0053] In some embodiments, (a) the target human protein is tyrosinase, and the alpha chain comprises the amino acid sequence as set forth in SEQ ID NO: 1; (b) the target human protein is MAGE-A1, and the alpha chain comprises the amino acid sequence as set forth in SEQ ID NO: 11; (c) the target human protein is MART1, and the alpha chain comprises the amino acid sequence as set forth in SEQ ID NO: 21; (d) the target human protein is MAGE-A3, and the alpha chain comprises the amino acid sequence as set forth in SEQ ID NO: 31; or (e) the target human protein is SSX2, and the alpha chain comprises the amino acid sequence as set forth in SEQ ID NO: 41.

[0054] In some embodiments, (a) the target human protein is tyrosinase and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 2; (b) the target human protein is MAGE-A1 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 12; (c) the target human protein is MART1 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 22; (d) the target human protein is MAGE-A3 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 32; or (e) the target human protein is SSX2 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 42.

[0055] Certain aspects of the present disclosure relate to a bispecific TCR comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises a TCR or antigen binding portion thereof disclosed herein or a recombinant TCR disclosed herein. In some embodiments, the first antigen binding domain comprises a single chain variable fragment (“scFv”). In some embodiments, the second antigen binding domain specifically binds to a protein expressed on the surface of a T cell. In some embodiments, the second antigen binding domain specifically binds to CD3. In some embodiments, the second antigen binding domain comprises a scFv. In some embodiments, the first antigen binding domain and the second antigen binding domain are linked or bound by a covalent bond. In some embodiments, the first antigen binding domain and the second antigen binding domain are linked by a peptide bond.

[0056] Certain aspects of the present disclosure relate to a cell comprising a nucleic acid molecule disclosed herein, a vector disclosed herein, a TCR disclosed herein, a recombinant TCR disclosed herein, or a bispecific TCR disclosed herein. In some embodiments, the cell further expresses CD3. In some embodiments, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, or an ILC cell.

[0057] Certain aspects of the present disclosure relate to a method of treating a cancer in a subject in need thereof comprising administering to the subject a cell disclosed herein. In some embodiments, the cancer is selected from the group consisting of melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), solid tumors of childhood, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary gland tumor, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of said cancers.

[0058] In some embodiments, the cancer is a relapsed or refractory cancer. In some embodiments, the cancer is a locally advanced cancer. In some embodiments, the cancer is an advanced cancer. In some embodiments, the cancer is a metastatic cancer.

[0059] In some embodiments, the cells are obtained from the subject. In some embodiments, the cells are obtained from a donor other than the subject. In some embodiments, the subject is pre-conditioned prior to administration of the cells. In some embodiments, pre-conditioning comprises administering to the subject a chemotherapy, a cytokine, a protein, a small molecule, or any combination thereof. In some embodiments, pre-conditioning comprises administering a leukocyte factor. In some embodiments, pre-conditioning comprises administering IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof. In some embodiments, pre-conditioning comprises administering a pre-conditioning agent selected from the group consisting of cyclophosphamide, fludarabine, vitamin C, an AKT inhibitor, ATRA, rapamycin, or any combination thereof. In some embodiments, pre-conditioning comprises administering cyclophosphamide, fludarabine, or both.

[0060] Certain aspects of the present disclosure relate to a method of engineering an antigen targeting cell, comprising transducing a cell collected from a subject in need of a T cell therapy with a nucleic acid molecule disclosed herein or a vector disclosed herein. In some embodiments, the antigen targeting cell further expresses CD3. In some embodiments, the cell is a T cell or a natural killer (NK) cell.

[0061] 057】Certain aspects of the present disclosure relate to an HLA class I molecule complexed with a peptide, wherein the HLA class I molecule comprises an alpha 1 domain, an alpha 2 domain, an alpha 3 domain, and a beta 2m, and wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 51, 52, 53, 54, 55, and any combination thereof. In some embodiments, the HLA class I molecule is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. In some embodiments, (a) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 51, and the HLA class I molecule is HLA-C; (b) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 52, and the HLA class I molecule is HLA-B; (c) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 53, and the HLA class I molecule is HLA-B; (d) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 54, and the HLA class I molecule is HLA-B; or (e) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 55, and the HLA class I molecule is HLA-A.

[0062] In some embodiments, (a) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 51, and the HLA class I molecule is HLA-C*05; (b) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 52, and the HLA class I molecule is HLA-B*07; (c) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 53, and the HLA class I molecule is HLA-B*18; (d) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 54, and the HLA class I molecule is HLA-B*18; or (e) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 55, and the HLA class I molecule is HLA-A*02.

[0063] In some embodiments, (a) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 51, and the HLA Class I molecule is selected from the group consisting of an HLA-C*05:01 allele, an HLA-C*05:03 allele, an HLA-C*05:04 allele, an HLA-C*05:05 allele, and an HLA-C*05:06 allele; (b) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 52, and the HLA Class I molecule is selected from the group consisting of an HLA-B*07:02 allele, an HLA-B*07:03 allele, an HLA-B*07:04 allele, an HLA-B*07:05 allele, and an HLA-B*07:06 allele; (c) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 53, and the HLA Class I molecule is selected from the group consisting of an HLA-B*18:01 allele, an HLA-B*18:02 allele, an HLA-B*18:03 allele, an HLA-B*18:04 allele, and an HLA-B*18:05 allele; (d) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 54, and the HLA Class I molecule is selected from the group consisting of an HLA-B*18:01 allele, an HLA-B*18:02 allele, an HLA-B*18:03 allele, an HLA-B*18:04 allele, and an HLA-B*18:05 allele; or (e) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 55, and the HLA Class I molecule is selected from the group consisting of an HLA-A*02:01 allele, an HLA-A*02:02 allele, an HLA-A*02:03 allele, an HLA-A*02:04 allele, and an HLA-A*02:05 allele.

[0064] In some embodiments, (a) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 51, and the HLA Class I molecule is HLA-C*05:01; (b) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 52, and the HLA Class I molecule is HLA-B*07:02; (c) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 53, and the HLA Class I molecule is HLA-B*18:01; (d) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 54, and the HLA Class I molecule is HLA-B*18:01; or (e) the peptide consists of the amino acid sequence set forth in SEQ ID NO: 55, and the HLA Class I molecule is HLA-A*02:01.

[0065] In some embodiments, the HLA Class I molecule is monomeric. In some embodiments, the HLA Class I molecule is dimeric. In some embodiments, the HLA Class I molecule is trimeric. In some embodiments, the HLA Class I molecule is tetrameric. In some embodiments, the HLA Class I molecule is pentameric.

[0066] Certain aspects of the present disclosure relate to an antigen presenting cell (APC) comprising an HLA Class I molecule disclosed herein. In some embodiments, the HLA Class I molecule is expressed on the surface of the APC.

[0067] Certain aspects of the present disclosure relate to a method of enriching a population of target T cells obtained from a human subject, comprising contacting the T cells with an HLA Class I molecule disclosed herein or an APC disclosed herein, wherein the enriched population of T cells comprises a higher number of T cells capable of binding to the HLA Class I molecule after the contacting relative to the number of T cells capable of binding to the HLA Class I molecule prior to the contacting.

[0068] Certain aspects of the present disclosure relate to a method of enriching a population of target T cells obtained from a human subject, comprising contacting the T cells in vitro with a peptide; wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 52, 53, 54, 55, and any combination thereof; wherein the enriched population of T cells comprises a higher number of T cells capable of targeting tumor cells after the contacting relative to the number of T cells capable of targeting tumor cells prior to the contacting. In some embodiments, the T cells obtained from a human subject are tumor infiltrating lymphocytes (TILs).

[0069] Certain aspects of the present disclosure relate to a method of treating a tumor in a subject in need thereof, comprising administering to the subject an enriched T cell disclosed herein.

[0070] Certain aspects of the present disclosure relate to a method of enhancing cytotoxic T cell-mediated targeting of cancer cells in a subject having a cancer, comprising administering to the subject a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 52, 53, 54, 55, and any combination thereof.

[0071] Certain aspects of the present disclosure relate to a cancer vaccine comprising a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 52, 53, 54, 55, and any combination thereof.

[0072] Certain aspects of the present disclosure relate to a method of selecting T cells capable of targeting tumor cells, comprising contacting an isolated population of T cells in vitro with a peptide, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 52, 53, 54, 55, and any combination thereof. In some embodiments, the T cells are tumor infiltrating lymphocytes (TILs). BRIEF DESCRIPTION OF DRAWINGS

[0073] Figures 1A-1D C*05:01 / Tyrosinase 460-468 Graphical representation of multimer staining. C*05:01 / Tyrosinase 460-468 peptide-pulsed C*05:01-artificial APCs stimulate TILs once. Shown are C*05:01 / Tyrosinase Figure 1A and Figure 1C 14 days after stimulation (day 14; Figure 1B and Figure 1D ) with C*05:01 / Tyrosinase 460-468 ( Figures 1A-1B ) or control C*05:01 / HIV rev 67-75 multimer Figures 1C-1D staining. Shown are percentages of C*05:01 / Tyrosinase + multimer + positive T cells.

[0074] Figure 2 Bar graphs showing functional evaluation of C*05:01 / Tyrosinase 460-468 multimer positive melanoma TILs. C*05:01 positive TILs produce IFN-γ in an HLA-C*05:01 restricted peptide specific manner. TILs were used as responder cells in an IFN-γ ELISPOT assay. C*05:01-artificial APCs pulsed with the indicated peptides were used as stimulator cells. HIV rev 67-75 peptide was used as control. Experiments were performed in triplicates and error bars depict standard deviation (SD). *P < 0.05.

[0075] Figures 3A-3I Graphical representation of positive staining of Jurkat 76 / CD8 cells transduced with C*05:01 / Tyrosinase 460-468 TCR with a cognate multimer. C*05:01 / Tyrosinase 460-468 TCR Figure 3B , Figure 3E and Figure 3H ) transduced Jurkat 76 / CD8 cells were stained with C*05:01 / Tyrosinase 460-468 multimer Figure 3B . C*05:01 / HIV rev67-75 Multimer Figure 3D , Figure 3E and Figure 3F ), C*05:01 / Unexchanged multimer Figure 3G , Figure 3H and Figure 3I ) and C*07:02 / MAGE-A1 289-297 TCR (clone CL2; Figure 3C , Figure 3F and Figure 3I ) and Jurkat 76 / CD8 Figure 3A , Figure 3D and Figure 3G ) not transduced with a TCR were used as controls. Multimer + CD8 + T cell percentages are shown.

[0076] Figures 4A-4D are graphical representations of positive staining of human primary T cells transduced with C*05:01 / Tyrosinase 460-468 TCR genes Figure 4B and Figure 4D ) by cognate multimers. Primary T cells transduced with C*05:01 / Tyrosinase 460-468 TCR were stained with C*05:01 / Tyrosinase 460-468 ( Figure 4B ) or C*05:01 / HIV rev 67-75 control multimers Figure 4D ). Non-transduced primary T cells were used as negative controls Figure 4A and Figure 4C ). Multimer + CD8 + T cell percentages are shown.

[0077] Figure 5 are bar graphs showing that human primary T cells transduced with C*05:01 / Tyrosinase 460-468 TCR genes strongly react with cognate peptides presented by the target class I molecule. Primary T cells transduced with C*05:01 / Tyrosinase 460-468 TCR genes or non-transduced primary T cells (x-axis) were used as responder cells in an IFN-γ ELISPOT assay. HLA-C*05:01 transduced T2 cells (T2-C*05:01) were generated. T2 or T2-C05:01 cells pulsed with Tyrosinase 460-468 or HIV rev 67-75 peptides (control) were used as stimulator cells. Experiments were performed in triplicate and error bars depict SD. ***P < 0.001.

[0078] Figure 6Ais a graph showing staining of melanoma TILs with B*07:02 / MAGE-A1 460-468 TCR gene transduced primary T cells recognize tumor cells. Primary T cells transduced with C*05:01 / Tyrosinase 460-468 TCR gene transduced primary T cells or untransduced primary T cells were used as responder cells in IFN-g ELISPOT analysis. Malme-3M, Me275 and MCF7 cells untransduced or transduced with HLA-C*05:01 or Tyrosinase were used as stimulator cells after treatment with 100 ng / ml IFNγ for 48 hours as indicated. Experiments were performed in triplicate and error bars depict SD. *P<0.05, **P<0.01, ***P<0.001.

[0079] Figures 7A-7D is a graph showing expression of endogenous or transduced Tyrosinase gene. Target cells were analyzed for expression of endogenous or transduced Tyrosinase gene by intracellular flow cytometry after staining with anti-Tyrosinase mAb (open curve) and isotype control (filled curve).

[0080] Figures 8A-8D is a graph showing expression of ΔNGFR in target cells transduced with ΔNGFR-tagged full-length HLA-C*05:01 gene Figure 8B and Figure 8D . Surface expression of ΔNGFR in target cells transduced with ΔNGFR-tagged full-length HLA-C*05:01 gene was analyzed by flow cytometry after staining with anti-NGFR mAb (open curve) and isotype control (filled curve). ΔNGFR alone was used as control Figure 8A and Figure 8C .

[0081] Figures 9A-9B is a graph showing staining of melanoma TILs with B*07:02 / MAGE-A1 289-297 multimer. Figure 9A showing staining of TILs with B*07:02 / MAGE-A1 289-297 multimer. B*07:02 / EBV EBNA3A 379-387 ( Figure 9B ) multimer was used as negative control. Percentage of CD8 + T cells + cells.

[0082] Figure 10 is a bar graph showing functional evaluation of B*07:02 / MAGE-A1 289-297 multimer positive melanoma TILs. TILs were stimulated with B*07:02 / MAGE-A1 289-297 ​- produced IFN-gamma in a specific manner. TIL were used as responder cells in IFN-gamma ELISPOT analysis. B*07:02 - artificial APCs pulsed with the indicated peptides were used as stimulator cells. HIV nef 128-137 Peptides were used as controls. Experiments were performed in triplicates and error bars depict standard deviation (SD). *P < 0.05.

[0083] Figures 11A-11I transduced with B*07:02 / MAGE-A1 289-297 TCR genes. Jurkat 76 / CD8 cells transduced with the cognate multimers are positively stained. Jurkat 76 / CD8 cells transduced with B*07:02 / MAGE-A1 289-297 TCR Figure 11B , Figure 11E and Figure 11H ) are stained with B*07:02 / MAGE-A1 289-297 multimers Figure 11B . Jurkat 76 / CD8 cells transduced with B*07:02 / NY-ESO-1 60-72 multimers Figure 11D , Figure 11E and Figure 11F ), B*07:02 / Unexchanged multimers Figure 11G , Figure 11H and Figure 11I ) and B*07:02 / NY-ESO-1 60-72 TCR Figure 11C , Figure 11F and Figure 11I ) and Jurkat 76 / CD8 cells not transduced with a TCR Figure 11A , Figure 11D and Figure 11G ) are used as controls. The percentage of multimer + CD8 + cells is shown.

[0084] Figures 12A-12D transduced with B*07:02 / MAGE-A1 289-297 TCR genes Figure 12B and Figure 12D ) are positively stained with the cognate multimers. Primary T cells transduced with B*07:02 / MAGE-A1 289-297 TCR are stained with B*07:02 / MAGE-A1 289-297 Figure 12B ) or B*07:02 / HIV nef 128-137 control multimers Figure 12D . Non-transduced primary T cells are used as negative controls Figure 12A and​Figure 12C ) is a bar graph showing multimer + CD8 + Percentages of T cells.

[0085] Figure 13 is a bar graph showing that human primary T cells transduced with B*07:02 / MAGE-A1 289-297 TCR genes strongly react with cognate peptides presented by target class I molecules. Human primary T cells transduced with B*07:02 / MAGE-A1 289-297 TCR genes or untransduced primary T cells (x-axis) were used as responder cells in IFN-γ ELISPOT analysis. HLA-B*07:02 transduced T2 cells (T2-B*07:02) were generated. T2 or T2-B*07:02 cells pulsed with MAGE-A1 289-297 or HIV nef 128-137 Peptides (control) were used as stimulator cells. Experiments were performed in triplicate and error bars depict SD. **P < 0.01.

[0086] Figure 14A is a bar graph showing that human primary T cells transduced with B*07:02 / MAGE-A1 289-297 TCR genes recognize tumor cells. Human primary T cells transduced with B*07:02 / MAGE-A1 289-297 TCR genes or untransduced primary T cells were used as responder cells in IFN-γ ELISPOT analysis. Me275, SL-MEL-37 and SK-MEL-21 cells untransduced or transduced with HLA-B*07:02 or MAGE-A1 were used as stimulator cells as indicated Figure 14B . Experiments were performed in triplicate and error bars depict SD. *P < 0.05, **P < 0.01.

[0087] Figures 15A-15D is a graph showing expression of MAGE-A1 derived from endogenous or transduced full-length genes. Target cells were analyzed for expression of MAGE-A1 derived from endogenous or transduced full-length genes via intracellular flow cytometry after staining with anti-MAGE-A1 mAb (open curve) and isotype control (filled curve).

[0088] Figures 16A-16D is a graph showing that T cells transduced with ΔNGFR-tagged full-length HLA-B*07:02 gene Figure 16B and Figure 16D) Graphical representation of the expression of ΔNGFR in transduced target cells. Surface expression of ΔNGFR in target cells transduced with the ΔNGFR-tagged full-length HLA-B*07:02 gene was analyzed by flow cytometry after staining with anti-NGFR mAb (open curve) and isotype control (filled curve). ΔNGFR alone was used as a control Figure 16A and Figure 16C ).

[0089] Figure 17 is a bar graph showing the number of B*18:01 / MART1 T cells in melanoma TIL. TIL were used as responder cells in IFN-γ ELISPOT analysis. B*18:01-artificial APCs pulsed with overlapping peptides covering the whole protein of MART1 were used as stimulator cells. TIL showed a positive response to two adjacent peptides with the consensus sequence 21 YTTAEEAAGIGILTV 35 of MART1 when stimulated with B*18:01-artificial APCs pulsed with MART1 -derived overlapping peptides (see also Table 5). Experiments were performed in triplicates and error bars depict SD. *P < 0.05, ***P < 0.001.

[0090] Figures 18A-18C is a graphical representation of B*18:01 / MART1 25-33 multimer staining of melanoma TIL. Figure 18B shows the staining of TIL with B*18:01 / MART1 25-33 multimer. B*18:01 / HIV gag 161-170 ( Figure 18C ) and B*18:01 / No exchange Figure 18A ) multimers were used as negative controls. The percentage of CD8 + T cells in multimer + positive melanoma TIL.

[0091] Figure 19 is a bar graph showing the functional evaluation of B*18:01 / MART1 25-33 multimer positive melanoma TIL. TIL produced IFN-γ in a B*18:01 / MART1 25-33 specific manner. TIL were used as responder cells in IFN-γ ELISPOT analysis. B*18:01-artificial APCs pulsed with the indicated peptides were used as stimulator cells. HIV gag 161-170 peptides were used as controls. Experiments were performed in triplicates and error bars depict standard deviation (SD). ***P < 0.001.

[0092] Figures 20A-20I ​Jurkat 76 / CD8 cells transduced with B*18:01 / MART1 25-33 TCR genes are shown. Jurkat 76 / CD8 cells transduced with B*18:01 / MART1 25-33 TCR Figure 20B , Figure 20E and Figure 20H ) are shown. Jurkat 76 / CD8 cells transduced with B*18:01 / MART1 25-33 multimers Figure 20B ) are shown. B*18:01 / MAGE-A3 167-176 multimers Figure 20D , Figure 20E and Figure 20F ), B*18:01 / MAGE-A3 167-176 TCR Figure 20C , Figure 20F and Figure 20I ) and B*18:01 / Untransposed multimers Figure 20G , Figure 20H and Figure 20I ) and Jurkat 76 / CD8 not transduced with TCR Figure 20A , Figure 20D and Figure 20G ) are used as controls. The percentage of multimer + CD8 + cells is shown.

[0093] Figures 21A-21D Jurkat 76 / CD8 cells transduced with B*18:01 / MART1 25-33 TCR genes Figure 21B and Figure 21D ) are shown. Jurkat 76 / CD8 cells transduced with B*18:01 / MART1 25-33 TCR are shown. Primary T cells transduced with B*18:01 / MART1 25-33 ( Figure 21B ) or B*18:01 / HIV gag 161-170 control multimers Figure 21D ) are shown. Non-transduced primary T cells are used as negative controls Figure 21A and Figure 21C ). The percentage of multimer + CD8 + T cells is shown.

[0094] Figure 22 Jurkat 76 / CD8 cells transduced with B*18:01 / MART1 25-33 TCR genes are shown. Jurkat 76 / CD8 cells transduced with B*18:01 / MART1 25-33Primary T cells transduced with TCR genes or untransduced primary T cells (x-axis) were used as responder cells in IFN-γ ELISPOT assays. HLA-B*18:01 transduced T2 cells (T2-B*18:01) were generated. T2 or T2-B*18:01 cells pulsed with MART1 25-33 or HIV gag 161-170 peptides (control) were used as stimulator cells. Experiments were performed in triplicate and error bars depict SD. **P < 0.01.

[0095] Figure 23A is a graph showing recognition of tumor cells by primary T cells transduced with B*18:01 / MART1 25-33 TCR genes. T2 cells transduced with B*18:01 / MART1 25-33 TCR genes or untransduced primary T cells were used as responder cells in IFN-γ ELISPOT assays. Malme-3M, SL-MEL-28 and A375 cells untransduced or transduced with HLA-B*18:01 or MART1 were used as stimulator cells as indicated Figure 23B . Experiments were performed in triplicate and error bars depict SD. *P < 0.05, **P < 0.01.

[0096] Figures 24A-24E is a graph showing expression of MART1 derived from endogenous or transduced full-length genes. Expression of MART1 derived from endogenous or transduced genes in target cells was analyzed by intracellular flow cytometry after staining with anti-MART1 mAb (open curve) and isotype control (filled curve).

[0097] Figures 25A-25F is a graph showing expression of ΔNGFR in target cells transduced with ΔNGFR-tagged full-length HLA-B*18:01 genes Figure 25B , Figure 25D and Figure 25F . Surface expression of ΔNGFR in target cells transduced with ΔNGFR-tagged full-length HLA-B*18:01 genes was analyzed by flow cytometry after staining with anti-NGFR mAb (open curve) and isotype control (filled curve). ΔNGFR alone was used as control Figure 25A , Figure 25C and Figure 25E .

[0098] Figures 26A-26C is a graph showing B*18:01 / MAGE-A3 167-176 multimer staining of melanoma TILs. T2 cells transduced with MAGE-A3 167-176Peptide pulse treatment with B*18:01-artificial APC stimulated TIL once. The results are shown before stimulation (day 0). Figure 26A ) and 14 days after stimulation (day 14; Figures 26B-26C ) of B*18:01 / MAGE-A3 167-176 ( Figures 26A-26B (or compare with B18:01 / HIV gag) 161-170 polymers ( Figure 26C Data on CD8 staining. (The data is shown.) + Multimers in T cells + Percentage of cells.

[0099] Figure 27 This shows B*18:01 / MAGE-A3 167-176 Bar graph of functional assessment of multimer-positive melanoma TILs. B*18:01-positive TILs produce IFN-γ in a peptide-specific manner restricted by HLA-B*18:01. (The last part, "using MAGE-A3," appears to be incomplete and requires further context.) 167-176 TILs stimulated with B*18:01-artificial APCs treated with peptide pulses were used as reaction cells in the IFN-γELISPOT assay. HLA-B*18:01-transduced T2 cells (T2-B*18:01) were generated. T2 or T2-B*18:01 cells treated with the indicated peptide pulses were used as stimulation cells. HIV gag 161-170 Peptides were used as controls. Experiments were performed in triplicate, and error bars were used to plot the standard deviation (SD). **P < 0.01, ***P < 0.001.

[0100] Figures 28A-28I Use B*18:01 / MAGE-A3 167-176 Illustration of Jurkat 76 / CD8 cells transduced with the TCR gene after homologous multimer positive staining. (Used B*18:01 / MAGE-A3) 167-176 TCR( Figure 28B , Figure 28E and Figure 28H Jurkat76 / CD8 cells transduced via B*18:01 / MAGE-A3 167-176 polymers ( Figure 28B Staining. B*18:01 / MART1 25-33 polymers ( Figure 28D , Figure 28E and Figure 28F B*18:01 / Unexchanged multimer ( Figure 28G , Figure 28H and Figure 28I ) and B*18:01 / MART1 25-33 TCR( Figure 28C ,Figure 28F and Figure 28I ) and Jurkat 76 / CD8 ( Figure 28A , Figure 28D and Figure 28G ) were used as controls. Multimer + CD8 + T cells are shown.

[0101] Figures 29A-29D is a bar graph showing human primary T cells transduced with B*18:01 / MAGE-A3 167-176 TCR gene Figure 29B and Figure 29D ) were transduced with B*18:01 / MAGE-A3 167-176 TCR. Primary T cells transduced with B*18:01 / MAGE-A3 167-176 ( Figure 29B ) or B18:01 / HIV gag 161-170 control multimers Figure 29D ) were stained. Non-transduced primary T cells were used as negative controls Figure 29A and Figure 29C ). Multimer + CD8 + T cells are shown.

[0102] Figure 30 is a bar graph showing human primary T cells transduced with B*18:01 / MAGE-A3 167-176 TCR gene strongly react with the cognate peptide presented by the target MHC class I molecule. Primary T cells transduced with B*18:01 / MAGE-A3 167-176 TCR gene or non-transduced primary T cells (x-axis) were used as responder cells in an IFN-γ ELISPOT assay. T2 cells pulsed with MAGE-A3 167-176 or HIV gag 161-170 peptides (control) were used as stimulator cells. Experiments were performed in triplicate and error bars depict SD. **P < 0.01.

[0103] Figure 31A is a bar graph showing primary T cells transduced with B*18:01 / MAGE-A3 167-176 TCR gene recognize tumor cells. Primary T cells transduced with B*18:01 / MAGE-A3 167-176 TCR gene or non-transduced primary T cells were used as responder cells in an IFN-γ ELISPOT assay. As indicated Figure 31B), SK-MEL-28 and HEK293T cells untransduced or transduced with HLA-B*07:02 and / or MAGE-A1 were used as stimulator cells. Experiments were performed in triplicate and error bars depict SD. *P<0.05, ***P<0.001.

[0104] Figure 32 is a graphical representation of expression of MAGE-A3 derived from endogenous or transduced full-length gene. Expression of MAGE-A3 derived from endogenous or transduced full-length gene in target cells was evaluated by Western blot analysis with anti-MAGE-A3 pAb. β-actin expression was used as positive control.

[0105] Figures 33A-33D is a graphical representation of expression of ΔNGFR in target cells transduced with full-length HLA-B*18:01 gene tagged with ΔNGFR Figure 33B and Figure 33D . Surface expression of ΔNGFR in target cells transduced with full-length HLA-B*18:01 gene tagged with ΔNGFR was analyzed by flow cytometry after staining with anti-NGFR mAb (open curve) and isotype control (filled curve). ΔNGFR alone was used as control Figure 33A and Figure 33C .

[0106] Figures 34A-34B is a graphical representation of A*02:01 / SSX2 41-49 multimer staining of melanoma TILs. Figure 34A shows staining of TILs by A*02:01 / SSX2 41-49 multimer. A*02:01 / HTLV-1tax 11-19 ( Figure 34B ) multimer was used as negative control. Percentage of multimer + positive CD8 + T cells in multimer 41-49 positive melanoma TILs.

[0107] Figure 35 is a bar graph showing functional evaluation of A*02:01 / SSX2 41-49 multimer positive melanoma TILs. TILs produced IFN-γ in an A*02:01 / SSX2 41-49 specific manner. TILs were used as responder cells in IFN-γ ELISPOT assay. T2 cells pulsed with the indicated peptides were used as stimulator cells. HTLV-1tax 11-19 peptide was used as control. Experiments were performed in triplicate and error bars depict standard deviation (SD). **P<0.01.

[0108] Figures 36A-36IJurkat 76 / CD8 cells transduced with A*02:01 / SSX2 41-49 TCR genes are shown. Jurkat 76 / CD8 cells transduced with A*02:01 / SSX2 41-49 TCR( Figure 36B , Figure 36E and Figure 36H ) are stained with A*02:01 / SSX2 41-49 multimers( Figure 36B ). A*02:01 / NY-ESO-1 157-165 multimers( Figure 36D , Figure 36E and Figure 36F ), A*02:01 / Untransposed multimers( Figure 36G , Figure 36H and Figure 36I ) and A*02:01 / NY-ESO-1 157- 165 TCR (pure 1G4LY; Figure 36C , Figure 36F and Figure 36I ) and Jurkat 76 / CD8 cells not transduced with TCRs( Figure 36A , Figure 36D and Figure 36G ) are used as controls. The percentage of multimer + CD8 + cells is shown.

[0109] Figures 37A-37D Jurkat 76 / CD8 cells transduced with A*02:01 / SSX2 41-49 TCR genes are shown. Jurkat 76 / CD8 cells transduced with A*02:01 / SSX2 41-49 TCR( Figure 37B and Figure 37D ) are stained with A*02:01 / SSX2 41-49 ( Figure 37B ) or A*02:01 / HTLV-1 tax 11-19 control multimers( Figure 37D ). Non-transduced primary T cells are used as negative controls( Figure 37A and Figure 37C ). The percentage of multimer + CD8 + T cells is shown.

[0110] Figure 38 Jurkat 76 / CD8 cells transduced with A*02:01 / SSX2 41-49Bar graph showing the strong response of TCR gene-transduced human primary T cells to homologous peptides presented by target class I molecules. (Using A*02:01 / SSX2) 41-49 Primary T cells transduced with the TCR gene or untransduced primary T cells (x-axis) were used as reactants in the IFN-γ ELISPOT assay. Using SSX2... 41-49 Or HTLV-1tax 11-19 T2 cells treated with peptide (control) pulses were used as stimulators. Experiments were performed in triplicate, and error bars were used to plot the standard deviation (SD). **P < 0.01.

[0111] Figure 39A This shows the use of A*02:01 / SSX2 41-49 Illustration of primary T cells transduced with the TCR gene recognizing tumor cells. (Using A*02:01 / SSX2) 41-49 Primary T cells transduced with the TCR gene or untransduced primary T cells were used as reactants in the IFN-γ ELISPOT assay. (As indicated) Figure 39B Untransduced or HLA-A*02:01 or SSX2-transduced SK-MEL-21, SL-MEL-37, and SK-MEL-28 cells were used as stimulating cells. Experiments were performed in triplicate, and error bars were used to plot SD. **P<0.01, ***P<0.001.

[0112] Figure 40 This is a diagram illustrating the expression of SSX2 derived from the endogenous or transduced full-length gene. The expression of SSX2 derived from the endogenous or transduced full-length gene in target cells was evaluated using Western blot analysis with anti-SSX2 pAb. β-actin expression was used as a positive control.

[0113] Figures 41A-41D This is a diagram illustrating the expression of HLA-A2 derived from the endogenous or transduced full-length HLA-A*02:01 gene. After staining with anti-HLA-A2 mAb (open curve) and isotype control (filled curve), the expression of HLA-A2 derived from the endogenous or transduced full-length HLA-A*02:01 in target cells was analyzed by flow cytometry. Detailed Implementation

[0114] The present disclosure relates to TCRs or antigen-binding portions thereof that specifically bind to an epitope on a target human protein selected from the group consisting of tyrosinase, MAGE-A1, MART1, MAGE-A3, and SSX2, nucleic acid molecules encoding the same, and cells comprising the TCRs or nucleic acid molecules. Some aspects of the present disclosure relate to methods of treating cancer in a subject in need thereof comprising administering to the subject a cell. Other aspects of the present disclosure relate to HLA class I molecules complexed with a peptide comprising a tyrosinase, MAGE-A1, MART1, MAGE-A3, or SSX2 epitope.

[0115] I. Terminology

[0116] To enable a better understanding of the present disclosure, certain terms are first defined. As used in this application, unless specifically identified otherwise as provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0117] It should be noted that the term "a" or "an" entity refers to one or more than one of that entity; for example, "a nucleotide sequence" should be understood as meaning one or more nucleotide sequences. As such, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0118] Also, the use of "and / or" in this document is to be taken as specific disclosure of each of the various components of the classes of components described. For example, "A and / or B" is to be taken as specific disclosure of each of the following: A; B; A and B. Likewise, the term "and / or" as used herein is intended to include each of the following aspects: A; B; A or B; A and B; A and / or B; B and / or A; A, B, and / or C; A, B, and C; A, B, or C; A, B, A and / or C; B, A, and / or C; C, A, and / or B; B or C; B and C; A, B, and / or C; and A, B, and C. Likewise, the term "and / or" as used herein is intended to include each of the following aspects: A; B; A or B; A and B; A and / or B; B and / or A; A, B, and / or C; A, B, and C; A, B, or C; A, B, A and / or C; B, A, and / or C; C, A, and / or B; B or C; B and C; A, B, and / or C; and A, B, and C.

[0119] The term "about" is used herein to refer to approximate, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries of the range by the equivalent of one unit in the lower and upper bounds of the range. Generally, the term "about" is used herein to modify a numerical value by varying the value upward or downward (higher or lower) by 10%.

[0120] It should be understood that, when aspects are described herein with language such as "comprises" or "consists of," it is intended to stand in openended opposition to language such as "consisting only of" or "consisting of." That is, each aspect described with openended language is also described with closedended language.

[0121] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the following publications are hereby incorporated by reference for their teachings of nomenclature used herein: the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed. 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed. 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press.

[0122] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino-to-carboxyl orientation. The headings provided herein are not limitations of various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined herein have meanings ascribed to them by reference to the specification as a whole and, as such, should not be interpreted to have meanings that are inconsistent with those ascribed to such terms by the specification as a whole.

[0123] “Administering” refers to the introduction of an agent entity into the body of a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenchymal embolism, for example by injection or infusion. As used herein, the phrase “parenteral administration” denotes modes of administration other than enteral, and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and sternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, e.g., oral administration. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, e.g., intranasal, vaginal, rectal, sublingual, or topical. Administration can also be performed, e.g., once, multiple times, and / or over one or more extended periods of time.

[0124] As used herein, the term "T cell receptor" (TCR) refers to a heteromeric cell surface receptor capable of specific interaction with a target antigen. As used herein, "TCR" includes, but is not limited to, naturally occurring and non-naturally occurring TCRs; full-length TCRs and antigen-binding portions thereof; chimeric TCRs; TCR fusion constructs; and synthetic TCRs. In humans, TCRs are expressed on the surface of T cells, and they are responsible for T cell recognition and targeting of antigen-presenting cells. Antigen-presenting cells (APCs) display fragments of foreign proteins (antigens) complexed with major histocompatibility complexes (MHCs) (also referred to herein as complexed with HLA molecules, e.g., HLA class 1 molecules). TCRs recognize and bind to the antigen:HLA complex and recruit CD3 (expressed by T cells), thereby activating the TCR. The activated TCR initiates downstream signaling and an immune response, including destruction of the EPC.

[0125] Generally, a TCR can comprise two chains, an alpha chain and a beta chain (or, less commonly, a gamma chain and a delta chain), which are interconnected by disulfide bonds. Each chain comprises a variable domain (an alpha chain variable domain and a beta chain variable domain) and a constant region (an alpha chain constant region and a beta chain constant region). The variable domain is located distal to the cell membrane, and the variable domain interacts with an antigen. The constant region is located proximal to the cell membrane. The TCR can further comprise a transmembrane region and a short cytoplasmic tail. As used herein, the term "constant region" encompasses the transmembrane region and cytoplasmic tail, if present, as well as the traditional "constant region."

[0126] The variable domain can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each alpha chain variable domain and beta chain variable domain comprises three CDRs and four FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Each variable domain contains a binding domain that interacts with an antigen. While all three CDRs on each chain are involved in antigen binding, CDR3 is considered the primary antigen binding region. CDR1 also interacts with the antigen, while CDR2 is thought to recognize the HLA complex primarily.

[0127] Unless otherwise specified, and unless the context indicates otherwise, the term "TCR" also includes an antigen-binding fragment or antigen-binding portion of any of the TCRs disclosed herein, and includes monovalent and bivalent fragments or portions and single chain TCRs. The term "TCR" is not limited to naturally occurring TCRs that bind to the surface of a T cell. As used herein, the term "TCR" further refers to a TCR described herein expressed on the surface of a cell other than a T cell (e.g., a cell that naturally expresses or is modified to express CD3, as described herein), or a TCR described herein that is dissociated from the cell membrane (e.g., an isolated TCR or a soluble TCR).

[0128] “Antigen binding molecule,” “portion of a TCR,” or “TCR fragment” refers to any portion of a TCR that is less than the whole. An antigen binding molecule can include antigenic complementarity determining regions (CDRs).

[0129] “Antigen” refers to any molecule, e.g., a peptide, that elicits an immune response or is capable of being bound by a TCR. As used herein, “epitope” refers to a portion of a polypeptide that elicits an immune response or is capable of being bound by a TCR. The immune response can involve antibody production or the activation of specific immunologically-competent cells, or both. Those skilled in the art will readily recognize that any large molecule, including virtually all proteins or peptides, can serve as an antigen. The antigen and / or epitope can be expressed endogenously, i.e., from genomic DNA, or can be expressed recombinantly. The antigen and / or epitope can be specific to certain tissues, such as cancer cells, or can be widely expressed. Additionally, fragments of larger molecules can serve as antigens. In one embodiment, the antigen is a tumor antigen. The epitope can exist in a longer polypeptide (e.g., a protein), or the epitope can exist as a fragment of a longer polypeptide. In some embodiments, the epitope is complexed with a major histocompatibility complex (MHC) (also referred to herein as complexed with an HLA molecule, e.g., an HLA class 1 molecule).

[0130] As used herein, “tyrosinase” or “TYR” (UniProtKB - P14679) refers to a copper-containing oxidase that plays a role in the formation of pigments such as melanin and other polyphenolic compounds. Tyrosinase catalyzes the initial and rate-limiting step in a reaction cascade that leads to the production of melanin from tyrosine. In addition to hydroxylating tyrosine to DOPA (3,4-dihydroxyphenylalanine), tyrosinase also catalyzes the oxidation of DOPA to DOPA-quinone, and possibly the oxidation of DHI (5,6-dihydroxyindole) to indole-5,6-quinone. Tyrosinase is expressed in the retina, skin, heart, aorta, mouth, and various other organs in the human body. A typical tyrosinase amino acid sequence (SEQ ID NO: 89) is shown in Table 1.

[0131] As used herein, “melanoma-associated antigen 1” or “MAGE-A1” (UniProtKB - P43355) refers to a tumor antigen that is expressed in many cancer types, including melanoma, head or neck squamous cell carcinoma, lung cancer, and breast cancer. MAGE-A1 is not expressed in normal tissues except for testis. MAGE-A1 is thought to be involved in transcriptional regulation through interaction with SNW1 and recruitment of histone deacetylase HDAC1. MAGE-A1 is thought to also inhibit notch intracellular domain (NICD) transactivation and can play a role in aspects of embryonic development and tumor transformation or tumor progression. A MAGE-A1 amino acid sequence (SEQ ID NO: 90) is shown in Table 1.

[0132] As used herein, “MART1,” “MART-1,” or “melanoma antigen recognized by T cells 1” (UniProtKB - Q16655) refers to an antigen involved in melanosome biogenesis by ensuring the stability of GPR143. MART1 plays an important role in the expression, stability, trafficking, and processing of the melanocyte protein PMEL, which is critical for the formation of stage II melanosomes. MART1 is expressed in melanoma cells, melanocytes, and the retina. The MART1 amino acid sequence (SEQ ID NO: 91) is shown in Table 1.

[0133] As used herein, “MAGE-A3” or “melanoma-associated antigen 3” (UniProtKB - P43357) refers to an antigen thought to enhance the ubiquitin ligase activity of RING-type zinc finger containing E3 ubiquitin-protein ligases. MAGE-A3 can also serve to enhance the ubiquitin ligase activity of TRIM28 and stimulate p53 / TP53 ubiquitination by TRIM28. MAGE-A3 is also thought to act by recruiting and / or stabilizing Ubl-conjugating enzymes (E2) at the E3:substrate complex. MAGE-A3 can also play a role in aspects of embryonic development and tumor transformation or tumor progression. In vitro expression of MAGE-A3 promotes cell viability in melanoma cell lines. MAGE-A3 is expressed in many cancer types, including melanoma, head or neck squamous cell carcinoma, lung cancer, and breast cancer. MAGE-A3 is not expressed in normal tissues except testis and placenta. The MAGE-A3 amino acid sequence (SEQ ID NO: 92) is shown in Table 1.

[0134] As used herein, “SSX2” or “proteins SSX2” (UniProtKB - Q16385) is an antigen expressed in rhabdomyosarcoma and fibrosarcoma cell lines. SSX2 is also expressed at high levels in testis and at low levels in thyroid. The function of SSX2 is not known, but it is postulated that SSX2 can act as a regulator of transcription. The SSX2 amino acid sequence (SEQ ID NO: 93) is shown in Table 1.

[0135] Table 1. Target Protein Amino Acid Sequences

[0136]

[0137]

[0138] As used herein, the term (HLA) refers to human leukocyte antigen. HLA genes encode the major histocompatibility complex (MHC) proteins of humans. MHC proteins are expressed on the surface of cells and are involved in the activation of immune responses. HLA class I genes encode MHC class I molecules that are expressed on the surface of cells in complex with peptide fragments (antigens) of self or non-self proteins. T cells that express TCRs and CD3 recognize the antigen:MHC class I complex and initiate an immune response that targets and destroys antigen-presenting cells that display non-self proteins.

[0139] As used herein, (HLA class I molecule) or (HLA class I molecule) refers to the protein product of a wild-type or variant HLA class I gene that encodes a MHC class I molecule. Thus, (HLA class I molecule) and (MHC class I molecule) are used interchangeably herein.

[0140] MHC class I molecules comprise two protein chains: an alpha chain and a beta2-microglobulin (b2m) chain. Human b2m is encoded by the B2M gene. The amino acid sequence of b2m is set forth in SEQ ID NO: 56 (Table 2). The alpha chain of MHC class I molecules is encoded by the HLA gene complex. The HLA complex is located within the 6p21.3 region of the short arm of human chromosome 6 and contains more than 220 different functional genes. HLA genes are highly variable, with over 20,000 HLA alleles and related alleles, including over 15,000 HLA class I alleles known in the art, which encode thousands of HLA proteins, including over 10,000 HLA class I proteins (see, e.g., hla.alleles.org, last accessed February 27, 2019). There are at least three genes in the HLA complex that encode MHC class I alpha chain proteins: HLA-A, HLA-B, and HLA-C. Additionally, HLA-E, HLA-F, and HLA-G encode proteins that are related to MHC class I molecules.

[0141] Table 2. Amino acid sequence of human b2m

[0142]

[0143] The term “autologous” refers to any material derived from the same individual, after which the material is reintroduced into the individual. For example, autologous T cell therapy includes administration of T cells isolated from the same subject to the subject. The term “allogeneic” refers to any material derived from one individual, after which the material is introduced into another individual of the same species. For example, allogeneic T cell transplantation includes administration of T cells obtained from a donor other than the subject to the subject.

[0144] "Cancer" refers to a broad group of various disorders characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in malignant tumors, which are masses of aberrant cells that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors. Examples of cancers that can be treated by the methods of the present application include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, and other white blood cell malignancies. In some embodiments, the methods of the present application can be used to reduce the size of a tumor derived from, for example, bone cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, malignant melanoma of the skin or eye, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, small bowel cancer, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of said cancers. The particular cancer can be responsive to chemotherapy or radiation therapy, or the cancer can be a refractory cancer. Refractory cancer refers to a cancer that is not affected by surgical intervention and that is initially unresponsive or becomes unresponsive over time to chemotherapy or radiation therapy.

[0145] As used herein, "anti-tumor effect" refers to a biological effect that can be expressed as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with the tumor. Anti-tumor effect can also refer to preventing tumor occurrence, e.g., a vaccine.

[0146] As used herein, the term "progression-free survival" can be abbreviated as PFS, and refers to the time from the date of treatment to the date of disease progression according to the revised IWG Response Criteria for Malignant Lymphoma or death from any cause.

[0147] As used herein, "disease progression" or "progressive disease" can be abbreviated as PD, and refers to the worsening of one or more symptoms associated with a particular disease. For example, disease progression in a subject afflicted with cancer can include an increase in the number or size of malignant lesions, metastasis of a tumor, and death.

[0148] As used herein, "duration of response" can be abbreviated as DOR, and refers to the period of time between a subject's first objective response to the date of documented disease progression or death according to the revised IWG Response Criteria for Malignant Lymphoma.

[0149] The term "overall survival" can be abbreviated as OS, and is defined as the time from the date of treatment to the date of death.

[0150] As used herein, “cytokine” refers to a non-antibody protein released by one cell in response to contact with a particular antigen, where the cytokine interacts with a second cell to mediate a response in the second cell. Cytokines can be expressed endogenously by a cell or administered to a subject. Cytokines can be released by immune cells, including macrophages, B cells, T cells, and mast cells to propagate an immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, proinflammatory cytokines, effectors, and acute phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, promote immune cell survival and proliferation, and proinflammatory cytokines can promote an inflammatory response. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) g. Examples of proinflammatory cytokines include, but are not limited to, IL-la, IL-lb, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-a, TNF-b, fibroblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0151] “Chemokine” is a type of cytokine that mediates chemotaxis or directed movement of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1a (MIP-1a, MIP-1a), MIP-1b (MIP-1b), IP-10, and thymus and activation-regulated chemokine (TARC or CCL17).

[0152] Other examples of analytes and cytokines of the present invention include, but are not limited to, chemokine (C-C motif) ligand (CCL) 1, CCL5, monocyte-specific chemokine 3 (MCP3 or CCL7), monocyte chemotactic protein 2 (MCP-2 or CCL8), CCL13, IL-1, IL-3, IL-9, IL-11, IL-12, IL-14, IL-17, IL-20, IL-21, granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), oncostatin M (OSM), CD154, lymphotoxin (LT) beta, 4-1BB ligand (4-1BBL), a proliferation-inducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-related ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L, TNF, and ApoL-related leukocyte-expressed ligand 1 (TALL-1), or TNF-related apoptosis-inducing ligand (TRAIL).

[0153] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes the decline of a disease, as evidenced by a lessening in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or a prevention of disease-caused impairment or disability. The ability of a therapeutic agent to promote the decline of a disease can be evaluated using a variety of methods known to the skilled artisan, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0154] As used herein, the term "lymphocyte" includes a natural killer (NK) cell, a T cell, or a B cell. NK cells are a type of cytotoxic (cell toxic) lymphocyte that represents a major component of the innate immune system. NK cells reject tumor and virus-infected cells. It acts through a process of apoptosis or programmed cell death. They are called "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without the involvement of antibodies). The T cell receptor (TCR) distinguishes T cells from other lymphocyte types. The thymus is a specialized organ of the immune system, primarily responsible for the maturation of T cells. There are six types of T cells, namely: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocyte, CTL, T killer cell, cytolytic T cell, CD8+ T cell, or killer T cell), memory T cells ((i) stem memory T cells (TSCM), (ii) central memory T cells (TCM), (iii) effector memory T cells (TEM), (iv) terminally differentiated effector memory T cells (TEMRA), and (v) mucosal-associated invariant T cells (MAIT)), regulatory T cells (Tregs), gamma delta T cells (gd T cells), and natural killer T cells (NKT cells). SCMCells (such as naive cells) are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Ra+, but they also express substantial amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and show many functional attributes that differ from memory cells); (ii) central memory T CM Cells express L-selectin and CCR7, they secrete IL-2 but not IFNy or IL-4, whereas (iii) effector memory T EM Cells do not express L-selectin or CCR7, but produce effector cytokines such as IFNy and IL-4), regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gd T cells. B cells, on the other hand, play a major role in humoral immunity (involvement of antibodies). B cells manufacture antibodies and antigens, and function as antigen presenting cells (APCs), and become memory B cells upon activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, from which they derive their name.

[0155] The term“genetically engineered” or“engineered” refers to a method of modifying the genome of a cell, including but not limited to, deletion of a coding or non-coding region or a portion thereof or insertion of a coding region or a portion thereof. In some embodiments, the modified cell is a lymphocyte, e.g., a T cell expressing CD3 or a modified cell that can be obtained from a patient or a donor. The cell can be modified to express an exogenous construct, such as, for example, a T cell receptor (TCR) disclosed herein, which is incorporated into the genome of the cell. In some embodiments, the cell is modified to express CD3.

[0156] “Immune response” refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules produced by any of these cells (including Abs, cytokines, and complement) that result in the selective targeting, binding, damaging, destruction, and / or elimination from a vertebrate’s body of: an invading pathogen, a cell or tissue infected with a pathogen, a cancerous or other abnormal cell, or, in the case of autoimmunity or pathological inflammation, a normal human cell or tissue.

[0157] The term "immunotherapy" refers to the treatment of a subject afflicted with, or at risk for acquiring or experiencing a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy can include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation.

[0158] Cells for use in the immunotherapies described herein can be from any source known in the art. For example, T cells can be differentiated in vitro from a population of hematopoietic stem cells, or T cells can be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In addition, T cells can be derived from one or more T cell lines available in the art. A number of techniques known to those of skill in the art can also be used, such as FICOLL TM T cells are isolated and / or apheresed from a unit of blood collected from a subject. Additional methods of isolating T cells for use in T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated by reference herein in its entirety. Immunotherapy can also include administering to a subject a modified cell, wherein the modified cell expresses CD3 and a TCR disclosed herein. In some embodiments, the modified cell is not a T cell.

[0159] As used herein, "patient" includes any human afflicted with a cancer (e.g., a lymphoma or a leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0160] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a compound comprising amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no maximum number of amino acids that can be included in a protein or peptide sequence. A polypeptide includes any peptide or protein comprising two or more amino acids linked to one another by peptide bonds. As used herein, the terms refer to short chains (which are also commonly referred to in the art as, e.g., peptides, oligopeptides, and oligomers) as well as longer chains (which are commonly referred to in the art as proteins). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide includes a natural, recombinant, synthetic, or a combination thereof.

[0161] As used herein, "stimulation" refers to the primary response induced by the binding of a stimulatory molecule to its cognate ligand, wherein the binding mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell, such as the T cell receptor (TCR) / CD3 complex, that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen presenting cell (e.g., aAPC, dendritic cell, B cell, and the like), can specifically bind to a stimulatory molecule on a T cell, thereby mediating a primary response of the T cell, including but not limited to activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands include, but are not limited to, MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0162] The terms "conditioning" and "pre-conditioning" are used interchangeably herein and indicate preparing a patient in need of a T cell therapy to a suitable state. As used herein, conditioning includes, but is not limited to, prior to a T cell therapy, reducing the number of endogenous lymphocytes, removing a cytokine sink, increasing serum levels of one or more homeostatic cytokines or proinflammatory factors, enhancing the effector function of T cells administered following conditioning, enhancing antigen presenting cell activation and / or availability, or any combination thereof. In one embodiment, "conditioning" comprises increasing serum levels of one or more cytokines, such as interleukin 7 (IL-7), interleukin 15 (IL-15), interleukin 10 (IL-10), interleukin 5 (IL-5), gamma-induced protein 10 (IP-10), interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1), placental growth factor (PLGF), C-reactive protein (CRP), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), or any combination thereof. In another embodiment, "conditioning" comprises increasing serum levels of IL-7, IL-15, IP-10, MCP-1, PLGF, CRP, or any combination thereof.

[0163] "Treatment" or "treating" of a subject refers to any type of intervention or process performed on a subject or administration of an active agent to a subject with the objective of reversing, alleviating, improving, inhibiting, slowing down or preventing the onset of the symptoms, complications or progression of a disease, or a biochemical indicator associated with the disease. In one embodiment, "treatment" includes partial remission. In another embodiment, "treatment" includes complete remission.

[0164] Use of the alternative (e.g.,“or”) should be understood to mean either one, but not both, of the alternatives. As used herein, the indefinite article“a” should be understood to refer to“one or more” of any stated or

[0165] The term“about” or“substantially” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example,“about” or“substantially” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively,“about” or“substantially” can mean ranges within 10% (i.e., ±10%). For example, about 3 mg can include any number between 2.7 mg and 3.3 mg (for 10%). Also, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to five-fold the amount. When a particular value or composition is provided in the application and claims, unless otherwise stated the meaning of“about” or“substantially” should be assumed to be within an acceptable error range of the particular value or composition.

[0166] Any concentration range, percentage range, ratio range, or integer range, described herein, should be interpreted as including any integer within the stated range and, when appropriate, fractions of the integers that are within the stated range.

[0167] Aspects of the application are described in more detail in the following subsections.

[0168] II. Compositions of the Disclosure

[0169] The present disclosure relates to T cell receptors (TCRs) or antigen binding portions thereof that specifically bind to an epitope on a target human protein selected from the group consisting of tyrosinase, MAGE-A1, MART1, MAGE-A3, and SSX2, nucleic acid molecules encoding the same, and cells comprising the TCR or nucleic acid molecule. Some aspects of the disclosure relate to methods of treating cancer in a subject in need thereof comprising administering to the subject a cell comprising a TCR described herein. Other aspects of the disclosure relate to the epitope of tyrosinase, MAGE-A1, MART1, MAGE-A3, or SSX2 bound by the TCR and an HLA class I molecule complexed with a peptide comprising the epitope of tyrosinase, MAGE-A1, MART1, MAGE-A3, or SSX2.

[0170] A T cell receptor or TCR is a molecule present on the surface of T cells or T lymphocytes that is responsible for recognizing a fragment of an antigen that is a peptide bound to a major histocompatibility complex (MHC) molecule. The binding between a TCR and an antigenic peptide has a relatively low affinity and is degenerate: that is, many TCRs recognize the same antigenic peptide and many antigenic peptides are recognized by the same TCR.

[0171] A TCR is composed of two different protein chains (i.e., it is a heterodimer). In humans, in 95% of T cells, the TCR is composed of an alpha (a) chain and a beta (b) chain (encoded by TRA and TRB, respectively), while in 5% of T cells, the TCR is composed of a gamma and delta (g / d) chain (encoded by TRG and TRD, respectively). This ratio varies during ontogeny and in diseased states, such as leukemia. It also differs between species. Orthologues of the four loci have been located in multiple species. Each locus can give rise to various polypeptides with constant and variable regions.

[0172] When a TCR engages an antigenic peptide and MHC (peptide / MHC), the T lymphocyte is activated through signal transduction, i.e., a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.

[0173] II.A. NUCLEIC ACID MOLECULES

[0174] Certain aspects of the present disclosure relate to nucleic acid molecules comprising (i) a first nucleotide sequence encoding a recombinant TCR or antigen binding portion thereof that specifically binds to a target human protein selected from the group consisting of tyrosinase, MAGE-A1, MART1, MAGE-A3, and SSX2; and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits expression of an endogenous TCR. In some embodiments, the second nucleotide sequence is a non-naturally occurring sequence. In other embodiments, the second nucleotide sequence is a synthetic nucleotide sequence. In other embodiments, the second nucleotide sequence comprises a sequence that targets a nucleotide sequence encoding an endogenous TCR. In some embodiments, the epitope specific TCR cross-competes for binding to the target human protein with a reference TCR. In some embodiments, the TCR binds to the same epitope or an overlapping epitope of the target human protein as the reference TCR.

[0175] In some embodiments, the reference TCR comprises an alpha chain and a beta chain; wherein the alpha chain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3; wherein the beta chain comprises a CDR1, a CDR2, and a CDR3; and wherein the reference TCR comprises an alpha chain CDR3 amino acid sequence selected from the amino acid sequences set forth in SEQ ID NO: 7, 17, 27, 37, and 47; and a beta chain CDR3 amino acid sequence selected from the amino acid sequences set forth in SEQ ID NO: 10, 20, 30, 40, and 50. In some embodiments, the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7, and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 17, and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27, and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 37, and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 47, and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0176] In some embodiments, the reference TCR comprises alpha chain CDR1, CDR2, and CDR3 sequences present in an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NO: 1, 11, 21, 31, and 41, and the reference TCR comprises beta chain CDR1, CDR2, and CDR3 sequences present in an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NO: 2, 12, 22, 32, and 42. In some embodiments, the reference TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 1, 11, 21, 31, or 41; and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 2, 12, 22, 32, or 42.

[0177] Table 3A. Tyrosinase alpha chain and beta chain TCR sequences

[0178]

[0179]

[0180] Table 3B. MAGE-A1 alpha and beta chain TCR sequences

[0181]

[0182]

[0183] Table 3C. MART1 alpha and beta chain TCR sequences

[0184]

[0185]

[0186]

[0187] Table 3D. MAGE-A3 alpha and beta chain TCR sequences

[0188]

[0189]

[0190] Table 3E. SSX2 alpha and beta chain TCR sequences

[0191]

[0192]

[0193]

[0194] II. A. 1. Epitope-specific TCRs

[0195] Certain aspects of the present disclosure relate to an epitope-specific TCR. In some embodiments, the epitope-specific TCR is encoded by a first nucleotide sequence described herein. In some embodiments, the epitope-specific TCR, e.g., encoded by the first nucleotide sequence, specifically binds to an epitope of a target human protein selected from the group consisting of tyrosinase, MAGE-A1, MART1, MAGE-A3, and SSX2. In some embodiments, the epitope-specific TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain and a beta chain, wherein the alpha chain comprises a variable domain comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3; and wherein the beta chain comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3.

[0196] II. A. 1. a. Anti-tyrosinase TCRs

[0197] In some embodiments, the epitope-specific TCR, e.g., encoded by the first nucleotide sequence, specifically binds to an epitope on human tyrosinase (“anti-tyrosinase TCR”) and the anti-tyrosinase TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 7 (CLVGDVEGSQGNLIF). In some embodiments, the anti-tyrosinase TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10 (CASSHHSGGIYNEQFF). In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain CDR1, wherein the alpha chain CDR1 of the anti-tyrosinase TCR comprises an amino acid sequence as set forth in SEQ ID NO: 5 (NIATNDY). In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain CDR1, wherein the beta chain CDR1 of the anti-tyrosinase TCR comprises an amino acid sequence as set forth in SEQ ID NO: 8 (MNHEY).

[0198] In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain CDR2, wherein the alpha chain CDR2 of the anti-tyrosinase TCR comprises an amino acid sequence as set forth in SEQ ID NO: 6 (GYKTK). In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain CDR2, wherein the beta chain CDR2 of the anti-tyrosinase TCR comprises an amino acid sequence as set forth in SEQ ID NO: 9 (SVGAGI).

[0199] In certain embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises: an alpha chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 5; an alpha chain CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 6; an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 7; a beta chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 8; a beta chain CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 9; and a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10.

[0200] In some embodiments, a non-CDR region in the alpha chain and / or beta chain of an epitope specific TCR, e.g., an anti-tyrosinase TCR, is further modified, e.g., a substitution or mutation of one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, or six amino acids, whereby the alpha chain and / or beta chain is not naturally occurring. In some embodiments, the substitution or mutation can improve the TCR described herein in various ways, e.g., binding affinity, binding specificity, stability, adhesion, or any combination thereof.

[0201] In some embodiments, the anti-tyrosinase TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the anti-tyrosinase TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 1, wherein the anti-tyrosinase TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 7. In some embodiments, the anti-tyrosinase TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain present in the alpha chain amino acid sequence set forth in SEQ ID NO: 1.

[0202] In some embodiments, the anti-tyrosinase TCR encoded by the first nucleotide sequence comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the anti-tyrosinase TCR encoded by the first nucleotide sequence comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 2, wherein the anti-tyrosinase TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, the anti-tyrosinase TCR encoded by the first nucleotide sequence comprises a beta chain variable domain present in the amino acid sequence set forth in SEQ ID NO: 2.

[0203] In some embodiments, the anti-tyrosinase TCR encoded by the first nucleotide sequence further comprises an alpha chain constant region, a beta chain constant region, or both an alpha chain constant region and a beta chain constant region. In some embodiments, the anti-tyrosinase TCR encoded by the first nucleotide sequence comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the anti-tyrosinase TCR encoded by the first nucleotide sequence comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 1, wherein the anti-tyrosinase TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 7. In some embodiments, the anti-tyrosinase TCR encoded by the first nucleotide sequence comprises an alpha chain constant region present in the alpha chain amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the anti-tyrosinase TCR encoded by the first nucleotide sequence further comprises an alpha constant region that is different from the endogenous, e.g., naturally occurring, constant region of the alpha chain. In some embodiments, the alpha chain constant region comprises an amino acid sequence that comprises at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the amino acid sequence of the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 1.

[0204] In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 2, wherein the anti-tyrosinase TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 10. In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region present in the beta chain amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, further comprises a beta constant region that is different from the endogenous, e.g., naturally occurring, constant region of the beta chain. In some embodiments, the beta chain constant region comprises an amino acid sequence that comprises at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 2.

[0205] In certain embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO: 1, wherein the anti-tyrosinase TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 7. In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 1.

[0206] In certain embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the beta chain amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the beta chain amino acid sequence set forth in SEQ ID NO: 2, wherein the anti-tyrosinase TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain comprising an amino acid sequence as set forth in SEQ ID NO: 2.

[0207] In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain constant region, a beta chain constant region, or both; wherein the alpha chain constant region, the beta chain constant region, or both comprise an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 substitutions within a target sequence relative to a corresponding amino acid sequence of an endogenous TCR. In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region, and wherein the beta chain comprises a constant region; wherein (i) the alpha chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to an alpha chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 1; and (ii) the beta chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to a beta chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0208] In some embodiments, the anti-tyrosinase TCR, e.g., encoded by the first nucleotide sequence, cross-competes for binding to human tyrosinase with a reference TCR. In some embodiments, the anti-tyrosinase TCR binds to the same epitope or an overlapping epitope of human tyrosinase as the reference TCR. In some embodiments, the reference TCR comprises an alpha chain and a beta chain, and the alpha chain of the reference TCR comprises an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the beta chain of the reference TCR comprises an amino acid sequence as set forth in SEQ ID NO: 2.

[0209] II.A.1.b. Anti-MAGE-A1 TCR

[0210] In some embodiments, the epitope-specific TCR, e.g., encoded by the first nucleotide sequence, specifically binds an epitope on human MAGE-A1 (“anti-MAGE-A1 TCR”) and the anti-MAGE-A1 TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 17 (CALSESYSGAGSYQLTF). In some embodiments, the anti-MAGE-A1 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 20 (CASSLASGSNQPQHF).

[0211] In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain CDR1, wherein the alpha chain CDR1 of the anti-MAGE-A1 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 15 (TRDTTYYL). In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain CDR1, wherein the beta chain CDR1 of the anti-MAGE-A1 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 18 (SEHNR).

[0212] In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain CDR2, wherein the alpha chain CDR2 of the anti-MAGE-A1 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 16 (RNSFDEQN). In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain CDR2, wherein the beta chain CDR2 of the anti-MAGE-A1 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 19 (FQNEAQ).

[0213] In certain embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises: an alpha chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 15; an alpha chain CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 16; an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 17; a beta chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 18; a beta chain CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 19; and a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 20.

[0214] In some embodiments, non-CDR regions in the alpha chain and / or beta chain of an epitope-specific TCR, e.g., an anti-MAGE-A1 TCR, are further modified, e.g., substitution or mutation of one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, or six amino acids, whereby the alpha chain and / or beta chain is not naturally occurring. In some embodiments, the substitution or mutation can improve the TCR described herein in various ways, e.g., binding affinity, binding specificity, stability, adhesion, or any combination thereof.

[0215] In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 11, wherein the anti-MAGE-A1 TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 17. In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain variable domain present in the alpha chain amino acid sequence set forth in SEQ ID NO: 11.

[0216] In some embodiments, the anti-MAGE-A2 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the anti-MAGE-A2 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 12, wherein the anti-MAGE-A2 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 20. In some embodiments, the anti-MAGE-A2 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain variable domain present in the beta chain amino acid sequence set forth in SEQ ID NO: 12.

[0217] In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, further comprises an alpha chain constant region, a beta chain constant region, or both an alpha chain constant region and a beta chain constant region. In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 11, wherein the anti-MAGE-A1 TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 17. In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises the alpha chain constant region present in the alpha chain amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, further comprises an alpha constant region that is different from an endogenous, e.g., naturally occurring, constant region of an alpha chain. In some embodiments, the alpha chain constant region comprises an amino acid sequence that is at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the amino acid sequence of the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 11.

[0218] In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 12, wherein the anti-MAGE-A1 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 20. In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region present in the beta chain amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, further comprises a beta constant region that is different from an endogenous, e.g., naturally occurring, constant region of the beta chain. In some embodiments, the beta chain constant region comprises an amino acid sequence that comprises at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 12.

[0219] In certain embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO: 11, wherein the anti-MAGE-A1 TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 17. In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 11.

[0220] In certain embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the beta chain amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the beta chain amino acid sequence set forth in SEQ ID NO: 12, wherein the anti-MAGE-A1 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain comprising an amino acid sequence as set forth in SEQ ID NO: 12.

[0221] In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain constant region, a beta chain constant region, or both; and wherein the alpha chain constant region, the beta chain constant region, or both comprise an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 substitutions within a target sequence relative to a corresponding amino acid sequence of an endogenous TCR. In some embodiments, the anti-MAGE-A1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region, and wherein the beta chain comprises a constant region; wherein (i) the alpha chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to an alpha chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 11; and (ii) the beta chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to a beta chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0222] In some embodiments, the anti-MAGE-A1 TCR cross-competes for binding to human MAGE-A1 with a reference TCR. In some embodiments, the anti-MAGE-A1 TCR binds to the same epitope or an overlapping epitope of human MAGE-A1 as the reference TCR. In some embodiments, the reference TCR comprises an alpha chain and a beta chain, and the alpha chain of the reference TCR comprises an amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, the beta chain of the reference TCR comprises an amino acid sequence as set forth in SEQ ID NO: 12.

[0223] II.A.1.c. Anti-MART1 TCR

[0224] In some embodiments, the epitope-specific TCR, e.g., encoded by the first nucleotide sequence, specifically binds an epitope on human MART1 (“anti-MART1 TCR”), and the anti-MART1 TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 27 (CAVYGGATNKLIF). In some embodiments, the anti-MART1 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 30 (CASSPHAGGVDEKLFF).

[0225] In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain CDR1, wherein the alpha chain CDR1 of the anti-MART1 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 25 (TSGFNG). In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain CDR1, wherein the beta chain CDR1 of the anti-MART1 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 28 (KGHSH).

[0226] In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain CDR2, wherein the alpha chain CDR2 of the anti-MART1 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 26 (NVLDGL). In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain CDR2, wherein the beta chain CDR2 of the anti-MART1 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 29 (LQKENI).

[0227] In certain embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises: an alpha chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 25; an alpha chain CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 26; an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 27; a beta chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 28; a beta chain CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 29; and a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 30.

[0228] In some embodiments, non-CDR regions in the alpha chain and / or beta chain of an epitope-specific TCR, such as an anti-MART1 TCR, are further modified, e.g., by substitution or mutation of one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, or six amino acids, whereby the alpha chain and / or beta chain is not naturally occurring. In some embodiments, the substitution or mutation can improve the TCR described herein in various ways, such as binding affinity, binding specificity, stability, avidity, or any combination thereof.

[0229] In some embodiments, the anti-MART1 TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the anti-MART1 TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 21, wherein the anti-MART1 TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 27. In some embodiments, the anti-MART1 TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain present in the alpha chain amino acid sequence set forth in SEQ ID NO: 21.

[0230] In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 22, wherein the anti-MART1 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 30. In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain variable domain present in the amino acid sequence set forth in SEQ ID NO: 22.

[0231] In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, further comprises an alpha chain constant region, a beta chain constant region, or both an alpha chain constant region and a beta chain constant region. In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 21, wherein the anti-MART1 TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 27. In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises the alpha chain constant region present in the alpha chain amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, further comprises an alpha constant region that is different from an endogenous, e.g., naturally occurring, constant region of an alpha chain. In some embodiments, the alpha chain constant region comprises an amino acid sequence that is at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the amino acid sequence of the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 21.

[0232] In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO:22. In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO:22, wherein the anti-MART1 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO:30. In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region present in the beta chain amino acid sequence set forth in SEQ ID NO:22. In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, further comprises a beta constant region that is different from an endogenous, e.g., naturally occurring, constant region of a beta chain. In some embodiments, the beta chain constant region comprises an amino acid sequence that comprises at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO:22.

[0233] In certain embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO:21. In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO:21, wherein the anti-MART1 TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO:27. In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO:21.

[0234] In certain embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the beta chain amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the beta chain amino acid sequence set forth in SEQ ID NO: 22, wherein the anti-MART1 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 30. In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain comprising an amino acid sequence as set forth in SEQ ID NO: 22.

[0235] In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain constant region, a beta chain constant region, or both; and wherein the alpha chain constant region, the beta chain constant region, or both comprise an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 substitutions within a target sequence relative to a corresponding amino acid sequence of an endogenous TCR. In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region, and wherein the beta chain comprises a constant region; wherein (i) the alpha chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to an alpha chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 21; and (ii) the beta chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to a beta chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 22.

[0236] In some embodiments, the anti-MART1 TCR, e.g., encoded by the first nucleotide sequence, cross-competes for binding to human MART1 with a reference TCR. In some embodiments, the anti-MART1 TCR binds to the same epitope or an overlapping epitope of human MART1 as the reference TCR. In some embodiments, the reference TCR comprises an alpha chain and a beta chain, and the alpha chain of the reference TCR comprises an amino acid sequence as set forth in SEQ ID NO: 21. In some embodiments, the beta chain of the reference TCR comprises an amino acid sequence as set forth in SEQ ID NO: 22.

[0237] II.A.1.d. Anti-MAGE-A3 TCR

[0238] In some embodiments, the epitope-specific TCR, e.g., encoded by the first nucleotide sequence, specifically binds an epitope on human MAGE-A3 (“anti-MAGE-A3 TCR”), and the anti-MAGE-A3 TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 37 (CAEVRSSASKIIF). In some embodiments, the anti-MAGE-A3 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 40 (CSANPRTTLYEQYF).

[0239] In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain CDR1, wherein the alpha chain CDR1 of the anti-MAGE-A3 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 35 (TRDTTYY). In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain CDR1, wherein the beta chain CDR1 of the anti-MAGE-A3 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 38 (DFQATT).

[0240] In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain CDR2, wherein the alpha chain CDR2 of the anti-MAGE-A3 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 36 (RNSFDEQN). In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain CDR2, wherein the beta chain CDR2 of the anti-MAGE-A3 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 39 (SNEGSKA).

[0241] In certain embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises: an alpha chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 35; an alpha chain CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 36; an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 37; a beta chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 38; a beta chain CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 39; and a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 40.

[0242] In some embodiments, non-CDR regions in the alpha chain and / or beta chain of an epitope-specific TCR, such as an anti-MAGE-A3 TCR, are further modified, e.g., by substitution or mutation of one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, or six amino acids, whereby the alpha chain and / or beta chain is not naturally occurring. In some embodiments, the substitution or mutation can improve the TCR described herein in various ways, e.g., binding affinity, binding specificity, stability, adhesion, or any combination thereof.

[0243] In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 31, wherein the anti-MAGE-A3 TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 37. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain variable domain present in the alpha chain amino acid sequence set forth in SEQ ID NO: 31.

[0244] In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 32, wherein the anti-MAGE-A3 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 40. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain variable domain present in the amino acid sequence set forth in SEQ ID NO: 32.

[0245] In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, further comprises an alpha chain constant region, a beta chain constant region, or both an alpha chain constant region and a beta chain constant region. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 31, wherein the anti-MAGE-A3 TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 37. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises the alpha chain constant region present in the alpha chain amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, further comprises an alpha constant region that is different from an endogenous, e.g., naturally occurring, constant region of an alpha chain. In some embodiments, the alpha chain constant region comprises an amino acid sequence that is at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the amino acid sequence of the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 31.

[0246] In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 32, wherein the anti-MAGE-A3 TCR comprises a beta chain CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 40. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region present in the beta chain amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, further comprises a beta constant region that is different from an endogenous, e.g., naturally occurring, constant region of the beta chain. In some embodiments, the beta chain constant region comprises an amino acid sequence that comprises at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 32.

[0247] In certain embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO: 31, wherein the anti-MAGE-A3 TCR comprises an alpha chain CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 37. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 31.

[0248] In certain embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the beta chain amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the beta chain amino acid sequence set forth in SEQ ID NO: 32, wherein the anti-MAGE-A3 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 40. In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain comprising an amino acid sequence as set forth in SEQ ID NO: 32.

[0249] In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain constant region, a beta chain constant region, or both; and wherein the alpha chain constant region, the beta chain constant region, or both comprise an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 substitutions within a target sequence relative to the corresponding amino acid sequence of an endogenous TCR.

[0250] In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region, and wherein the beta chain comprises a constant region; wherein (i) the alpha chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to an alpha chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 31; and (ii) the beta chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to a beta chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 32.

[0251] In some embodiments, the anti-MAGE-A3 TCR, e.g., encoded by the first nucleotide sequence, cross-competes for binding to human MAGE-A3 with a reference TCR. In some embodiments, the anti-MAGE-A3 TCR binds to the same epitope or an overlapping epitope of human MAGE-A3 as the reference TCR. In some embodiments, the reference TCR comprises an alpha chain and a beta chain, and the alpha chain of the reference TCR comprises an amino acid sequence as set forth in SEQ ID NO: 31. In some embodiments, the beta chain of the reference TCR comprises an amino acid sequence as set forth in SEQ ID NO: 32.

[0252] II.A.1.e. Anti-SSX2 TCR

[0253] In some embodiments, the epitope-specific TCR, e.g., encoded by the first nucleotide sequence, specifically binds to an epitope on human SSX2 (“anti-SSX2 TCR”), and the anti-SSX2 TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 47 (CAVEPMEYGNKLVF). In some embodiments, the anti-SSX2 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 50 (CASSALFSGANVLTF).

[0254] In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain CDR1, wherein the alpha chain CDR1 of the anti-SSX2 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 45 (DSAIYN). In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain CDR1, wherein the beta chain CDR1 of the anti-SSX2 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 48 (LNHDA).

[0255] In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain CDR2, wherein the alpha chain CDR2 of the anti-SSX2 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 46 (IQSSQRE). In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain CDR2, wherein the beta chain CDR2 of the anti-SSX2 TCR comprises an amino acid sequence as set forth in SEQ ID NO: 49 (SQIVND).

[0256] In certain embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 45; an alpha chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46; an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47; a beta chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 48; a beta chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 49; and a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50.

[0257] In some embodiments, non-CDR regions in the alpha chain and / or beta chain of an epitope specific TCR, e.g., an anti-SSX2 TCR, are further modified, e.g., substitution or mutation of one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, or six amino acids, whereby the alpha chain and / or beta chain are not naturally occurring. In some embodiments, the substitution or mutation can improve the TCR described herein in various ways, e.g., binding affinity, binding specificity, stability, avidity, or any combination thereof.

[0258] In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 41, wherein the anti-SSX2 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain variable domain present in the alpha chain amino acid sequence set forth in SEQ ID NO: 41.

[0259] In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the variable domain of the beta chain amino acid sequence set forth in SEQ ID NO:42. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the variable domain of the beta chain amino acid sequence set forth in SEQ ID NO:42, wherein the anti-SSX2 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO:50. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain variable domain present in the amino acid sequence set forth in SEQ ID NO:42.

[0260] In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, further comprises an alpha chain constant region, a beta chain constant region, or both an alpha chain constant region and a beta chain constant region. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 41, wherein the anti-SSX2 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 7. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain constant region present in the alpha chain amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, further comprises an alpha constant region that is different from an endogenous, e.g., naturally occurring, constant region of an alpha chain. In some embodiments, the alpha chain constant region comprises an amino acid sequence that is at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the amino acid sequence of the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 41.

[0261] In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO:42. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO:42, wherein the anti-SSX2 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO:50. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain constant region present in the beta chain amino acid sequence set forth in SEQ ID NO:42. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, further comprises a beta constant region that is different from an endogenous, e.g., naturally occurring, constant region of the beta chain. In some embodiments, the beta chain constant region comprises an amino acid sequence that comprises at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO:42.

[0262] In certain embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO:41. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO:41, wherein the anti-SSX2 TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO:47. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO:41.

[0263] In certain embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the beta chain amino acid sequence set forth in SEQ ID NO:42. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the beta chain amino acid sequence set forth in SEQ ID NO:42, wherein the anti-SSX2 TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO:50. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises a beta chain comprising an amino acid sequence as set forth in SEQ ID NO:42.

[0264] In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain constant region, a beta chain constant region, or both; and wherein the alpha chain constant region, the beta chain constant region, or both comprise an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 substitutions within a target sequence relative to a corresponding amino acid sequence of an endogenous TCR. In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region, and wherein the beta chain comprises a constant region; wherein (i) the alpha chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to an alpha chain constant region comprising the amino acid sequence set forth in SEQ ID NO:41; and (ii) the beta chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to a beta chain constant region comprising the amino acid sequence set forth in SEQ ID NO:42.

[0265] In some embodiments, the anti-SSX2 TCR, e.g., encoded by the first nucleotide sequence, cross-competes for binding to human SSX2 with a reference TCR. In some embodiments, the anti-SSX2 TCR binds to the same epitope or an overlapping epitope of human SSX2 as the reference TCR. In some embodiments, the reference TCR comprises an alpha chain and a beta chain, and the alpha chain of the reference TCR comprises an amino acid sequence as set forth in SEQ ID NO:41. In some embodiments, the beta chain of the reference TCR comprises an amino acid sequence as set forth in SEQ ID NO:42.

[0266] II.A.2 Second Nucleotide Sequence

[0267] The second nucleotide sequence of the nucleic acid molecule disclosed herein can be any sequence or can encode any polypeptide that is capable of inhibiting the expression of an endogenous TCR. In some embodiments, the second nucleotide sequence is one or more siRNAs. In some embodiments, the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of an endogenous TCR. In certain embodiments, the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of a wild-type human TCR. In some embodiments, the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of an alpha chain of a wild-type TCR. In some embodiments, the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of a beta chain of a wild-type TCR. In some embodiments, the one or more siRNAs comprise (i) one or more siRNAs that are complementary to a target sequence within a nucleotide sequence encoding a constant region of an alpha chain of a wild-type TCR and (ii) one or more siRNAs that are complementary to a target sequence within a nucleotide sequence encoding a constant region of a beta chain of a wild-type TCR.

[0268] In some embodiments, the one or more siRNAs comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 57-60 (Table 4). In some embodiments, the second nucleotide sequence of the nucleic acid molecule encodes one or more siRNAs, wherein the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of an alpha chain of a wild-type TCR, and wherein the one or more siRNAs comprise the nucleic acid sequences set forth in SEQ ID NOs: 57 and 58.

[0269] Table 4. siRNA Sequences

[0270]

[0271]

[0272] In some embodiments, the second nucleotide sequence of the nucleic acid molecule encodes one or more siRNAs, wherein the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of a beta chain of a wild-type TCR, and wherein the one or more siRNAs comprise the nucleic acid sequences set forth in SEQ ID NOs: 59 and 60. In some embodiments, the second nucleotide sequence of the nucleic acid molecule encodes one or more siRNAs, wherein the one or more siRNAs comprise (i) one or more siRNAs complementary to a target sequence within a nucleotide sequence encoding a constant region of an alpha chain of a wild-type TCR, wherein the one or more siRNAs comprise the nucleic acid sequences set forth in SEQ ID NOs: 57 and 58; and (ii) one or more siRNAs complementary to a target sequence within a nucleotide sequence encoding a constant region of a beta chain of a wild-type TCR, wherein the one or more siRNAs comprise the nucleic acid sequences set forth in SEQ ID NOs: 59 and 60.

[0273] In some embodiments, the second nucleotide sequence of the nucleic acid molecule comprises SEQ ID NOs: 57-60. In some embodiments, the second nucleotide sequence comprises SEQ ID NOs: 57-60, wherein one or more of SEQ ID NOs: 57-60 is separated by one or more nucleic acid that does not encode an siRNA. In certain embodiments, the one or more siRNAs are selected from the siRNAs disclosed in U.S. Pub. No. 2010 / 0273213 Al, which is incorporated by reference in its entirety.

[0274] In some embodiments, the second nucleotide sequence of the nucleic acid molecule encodes a protein, wherein the protein is capable of inhibiting expression of an endogenous, e.g., wild-type TCR. In some embodiments, the second nucleotide sequence encodes Cas9.

[0275] II.A.3 Vectors

[0276] Certain aspects of the present disclosure relate to vectors comprising the nucleic acid molecules disclosed herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a viral particle or virus. In some embodiments, the vector is a mammalian vector. In some embodiments, the vector is a bacterial vector.

[0277] In certain embodiments, the vector is a retroviral vector. In some embodiments, the vector is selected from the group consisting of an adenoviral vector, a lentivirus, a Sendai virus, a baculoviral vector, an Epstein Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, and an adeno-associated virus (AAV) vector. In particular embodiments, the vector is an AAV vector. In some embodiments, the vector is a lentivirus. In particular embodiments, the vector is an AAV vector. In some embodiments, the vector is a Sendai virus. In some embodiments, the vector is a hybrid vector. Examples of hybrid vectors that can be used in the present disclosure can be found in Huang and Kamihira, Biotechnol. Adv. 31(2):208-23 (2103), which is incorporated by reference in its entirety.

[0278] II.B. Recombinant T Cell Receptors (TCRs)

[0279] Certain aspects of the present disclosure relate to a recombinant T cell receptor (TCR) or antigen binding portion thereof that specifically binds to a target human protein selected from the group consisting of tyrosinase, MAGE-A1, MART1, MAGE-A3, and SSX2. In some embodiments, the antigen-specific TCR is encoded by a nucleic acid molecule disclosed herein.

[0280] The epitope-specific TCR can be selected from (i) any epitope-specific TCR disclosed herein, e.g., the TCRs disclosed in Sections IIA.1.a. through IIA.1.e. above, and (ii) any TCR that cross-competes for binding to the target human protein with a reference antibody selected from any epitope-specific TCR disclosed herein, e.g., the TCRs disclosed in Sections IIA.1.a. through IIA.1.e. above.

[0281] In certain embodiments, the epitope-specific TCR is an anti-tyrosinase TCR disclosed herein. In certain embodiments, the anti-tyrosinase TCR comprises an alpha chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5; an alpha chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6; an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7; a beta chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8; a beta chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9; and a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0282] In certain embodiments, the epitope-specific TCR is an anti-MAGE-A1 TCR disclosed herein. In certain embodiments, the anti-MAGE-A1 TCR comprises an alpha chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15; an alpha chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16; an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; a beta chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18; a beta chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19; and a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20.

[0283] In certain embodiments, the epitope-specific TCR is an anti-MART1 TCR disclosed herein. In certain embodiments, the anti-MART1 TCR comprises an alpha chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25; an alpha chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26; an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; a beta chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28; a beta chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29; and a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0284] In certain embodiments, the epitope-specific TCR is an anti-MAGE-A3 TCR disclosed herein. In certain embodiments, the anti-MAGE-A3 TCR comprises an alpha chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35; an alpha chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36; an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37; a beta chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38; a beta chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39; and a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40.

[0285] In certain embodiments, the epitope-specific TCR is an anti-SSX2 TCR disclosed herein. In certain embodiments, the anti-SSX2 TCR comprises an alpha chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 45; an alpha chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46; an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47; a beta chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 48; a beta chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 49; and a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50.

[0286] II.B.3. Bispecific T cell receptors (TCRs)

[0287] Certain aspects of the present disclosure relate to a bispecific TCR comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises a TCR or antigen binding portion thereof disclosed herein. In some embodiments, the bispecific TCR comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises an anti-tyrosinase TCR or antigen binding portion thereof disclosed herein. In some embodiments, the bispecific TCR comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises an anti-MAGE-A1 TCR or antigen binding portion thereof disclosed herein. In some embodiments, the bispecific TCR comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises an anti-MART1 TCR or antigen binding portion thereof disclosed herein. In some embodiments, the bispecific TCR comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises an anti-MAGE-A3 TCR or antigen binding portion thereof disclosed herein. In some embodiments, the bispecific TCR comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises an anti-SSX2 TCR or antigen binding portion thereof disclosed herein. In some embodiments, the first antigen binding domain comprises a single chain variable fragment (“scFv”).

[0288] In some embodiments, the second antigen binding domain specifically binds to a protein expressed on the surface of a T cell. Any protein expressed on the surface of a T cell can be targeted by the bispecific antibodies disclosed herein. In certain embodiments, the protein expressed on the surface of a T cell is not expressed by other cells. In some embodiments, the protein expressed on the surface of a T cell is expressed on the surface of one or more other human immune cells. In some embodiments, the protein expressed on the surface of a T cell is expressed on the surface of one or more other human immune cells, but it is not expressed on the surface of a human non-immune cell. In some embodiments, the second antigen binding domain specifically binds to a protein expressed on the surface of a T cell selected from the group consisting of CD3, CD2, CD5, CD6, CD8, CD11a (LFA-1 alpha), CD43, CD45, and CD53. In certain embodiments, the second antigen binding domain specifically binds to CD3. In some embodiments, the second antigen binding domain comprises a scFv.

[0289] In some embodiments, the first and second antigen binding domains are linked or bound by a covalent bond. In some embodiments, the first and second antigen binding domains are linked by a peptide bond.

[0290] II.C. Epitope

[0291] In some embodiments, the antigen-specific TCR binds to the same epitope as the reference TCR. In some embodiments, the antigen-specific TCR specifically binds to human tyrosinase (“anti-tyrosinase TCR”), wherein the anti-tyrosinase TCR binds to an epitope of tyrosinase comprising the amino acid sequence set forth in SEQ ID NO: 51 (FQDYIKSYL).

[0292] In some embodiments, the anti-tyrosinase TCR binds to an epitope of tyrosinase consisting of the amino acid sequence as set forth in SEQ ID NO: 51. In some embodiments, the epitope comprises amino acid residues 460-468 of tyrosinase (SEQ ID NO: 89), e.g., “tyrosinase 460-468 ”. In some embodiments, the epitope consists of amino acid residues 460-468 of tyrosinase (SEQ ID NO: 89), e.g., “tyrosinase 460-468 ”.

[0293] In some embodiments, the antigen-specific TCR specifically binds to human MAGE-A1 ("anti-MAGE-A1 TCR"), wherein the anti-MAGE-A1 TCR binds to an epitope of MAGE-A1 comprising the amino acid sequence set forth in SEQ ID NO: 52 (RVRFFFPSL). In some embodiments, the anti-MAGE-A1 TCR binds to an epitope of MAGE-A1 consisting of the amino acid sequence as set forth in SEQ ID NO: 52. In some embodiments, the epitope comprises amino acid residues 289-297 of MAGE-A1 (SEQ ID NO: 90), e.g., "MAGE-A1 289-297 ". In some embodiments, the epitope consists of amino acid residues 289-297 of MAGE-A1 (SEQ ID NO: 90), e.g., "MAGE-A1 289-297 ".

[0294] In some embodiments, the antigen-specific TCR specifically binds to human MART1 ("anti-MART1 TCR"), wherein the anti-MART1 TCR binds to an epitope of MART1 comprising the amino acid sequence set forth in SEQ ID NO: 53 (EEAAGIGIL). In some embodiments, the anti-MART1 TCR binds to an epitope of MART1 consisting of the amino acid sequence as set forth in SEQ ID NO: 53. In some embodiments, the epitope comprises amino acid residues 25-33 of MART1 (SEQ ID NO: 91), e.g., "MART1 25-33 ". In some embodiments, the epitope consists of amino acid residues 25-33 of MART1 (SEQ ID NO: 91), e.g., "MART1 25-33 ".

[0295] In some embodiments, the antigen-specific TCR specifically binds to human MAGE-A3 ("anti-MAGE-A3 TCR"), wherein the anti-MAGE-A3 TCR binds to an epitope of MAGE-A3 comprising the amino acid sequence set forth in SEQ ID NO: 54 (MEVDPIGHLY). In some embodiments, the anti-MAGE-A3 TCR binds to an epitope of MAGE-A3 consisting of the amino acid sequence as set forth in SEQ ID NO: 54. In some embodiments, the epitope comprises amino acid residues 167-176 of MAGE-A3 (SEQ ID NO: 92), e.g., "MAGE-A3 167-176 ". In some embodiments, the epitope consists of amino acid residues 167-176 of MAGE-A3 (SEQ ID NO: 92), e.g., "MAGE-A3167-176

[0296] In some embodiments, the antigen-specific TCR specifically binds to human SSX2 ("anti-SSX2 TCR"), wherein the anti-SSX2 TCR binds to an epitope of SSX2 comprising the amino acid sequence set forth in SEQ ID NO: 55 (KASEKIFYV). In some embodiments, the anti-SSX2 TCR binds to an epitope of SSX2 consisting of the amino acid sequence as set forth in SEQ ID NO: 55. In some embodiments, the epitope comprises amino acid residues 41-49 of SSX2 (SEQ ID NO: 93), e.g., "SSX2 41-49 ". In some embodiments, the epitope consists of amino acid residues 41-49 of SSX2 (SEQ ID NO: 93), e.g., "SSX2 41-49

[0297] II.D.HLA Class I Molecules

[0298] Certain aspects of the present disclosure relate to a complex comprising an HLA Class I molecule and an epitope disclosed herein. The HLA Class I molecule can be any HLA Class I molecule known in the art. In some embodiments, the HLA Class I molecule is selected from HLA-A, HLA-B, and HLA-C alleles. In some embodiments, the HLA Class I molecule is selected from HLA-E, HLA-F, and HLA-G alleles. In certain embodiments, the HLA Class I molecule is an HLA-A allele. In certain embodiments, the HLA Class I molecule is an HLA-B allele. In certain embodiments, the HLA Class I molecule is an HLA-C allele.

[0299] Many HLA-A, HLA-B, and HLA-C alleles are known in the art, and any known allele can be used in the present disclosure. An updated list of HLA alleles is available at hla.alleles.org / (last accessed on February 27, 2019).

[0300] II.D.1.HLA-A Alleles and Complexes Thereof

[0301] Certain aspects of the present disclosure relate to a complex comprising an HLA Class I molecule and an epitope, wherein the HLA Class I molecule is an HLA-A allele, and wherein the epitope is an SSX2 epitope disclosed herein. In certain embodiments, the SSX2 epitope comprises, consists of, or consists essentially of SEQ ID NO: 55.

[0302] ​​In some embodiments, the HLA-A allele is selected from HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-A*23, HLA-A*24, HLA-A*25, HLA-A*26, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*32, HLA-A*33, HLA-A*34, HLA-A*36, HLA-A*43, HLA-A*66, HLA-A*68, HLA-A*69, HLA-A*74, and HLA-A*80. In certain embodiments, the HLA-A allele is an HLA-A*02 allele.

[0303] In certain embodiments, the complex comprises an HLA-A*02 allele and an SSX2 epitope disclosed herein, e.g., an epitope comprising, consisting of, or consisting essentially of SEQ ID NO: 55. In certain embodiments, the HLA-A allele is an HLA-A*02:01 allele. In particular embodiments, the complex comprises an HLA-A*02:01 allele and an SSX2 epitope comprising, consisting of, or consisting essentially of SEQ ID NO: 55.

[0304] In certain embodiments, the HLA-A allele is selected from the group consisting of HLA-A*02:01:01:01, HLA-A*02:01:01:02L, HLA-A*02:01:01:03, HLA-A*02:01:01:04, HLA-A*02:01:01:05, HLA-A*02:01:01:06, HLA-A*02:01:01:07, HLA-A*02:01:01:08, HLA-A*02:01:01:09, HLA-A*02:01:01:10, HLA-A*02:01:01:11, HLA-A*02:01:01:12, HLA-A*02:01:01:13, HLA-A*02:01:01:14, HLA-A*02:01:01:15, HLA-A*02:01:01:16, HLA-A*02:01:01:17, HLA-A*02:01:01:18, HLA-A*02:01:01:19, HLA-A*02:01:01:20, HLA-A*02:01:01:21, HLA-A*02:01:01:22, HLA-A*02:01:01:23, HLA-A*02:01:01:24, HLA-A*02:01:01:25, HLA-A*02:01:01:26, HLA-A*02:01:01:27, HLA-A*02:01:01:28, HLA-A*02:01:01:29, HLA-A*02:01:01:30, HLA-A*02:01:01:31, HLA-A*02:01:01:32, HLA-A*02:01:01:33, HLA-A*02:01:01:34, HLA-A*02:01:01:35, HLA-A*02:01:01:36, HLA-A*02:01:01:37, HLA-A*02:01:01:38, HLA-A*02:01:01:39, HLA-A*02:01:01:40, HLA-A*02:01:01:41, HLA-A*02:01:01:42, HLA-A*02:01:01:43, HLA-A*02:01:01:44, HLA-A*02:01:01:45, HLA-A*02:01:01:46, HLA-A*02:01:01:47, HLA-A*02:01:01:48, HLA-A*02:01:01:49, HLA-A*02:01:01:50, HLA-A*02:01:01:51, HLA-A*02:01:01:52, HLA-A*02:01:01:53, HLA-A*02:01:01:54,HLA-A*02:01:01:55, HLA-A*02:01:02, HLA-A*02:01:03, HLA-A*02:01:04, HLA-A*02:01:05, HLA-A*02:01:06, HLA-A*02:01:07, HLA-A*02:01:08, HLA-A*02:01:09, HLA-A*02:01:10, HLA-A*02:01:100, HLA-A*02:01:101, HLA-A*02:01:102, HLA-A*02:01:103, HLA-A*02:01:104, HLA-A*02:01:105, HLA-A*02:01:106, HLA-A*02:01:107, HLA-A*02:01:108, HLA-A*02:01:109, HLA-A*02:01:11, HLA-A*02:01:110, HLA-A*02:01:111, HLA-A*02:01:112, HLA-A*02:01:113, HLA-A*02:01:114, HLA-A*02:01:115, HLA-A*02:01:116, HLA-A*02:01:117, HLA-A*02:01:118, HLA-A*02:01:119, HLA-A*02:01:12, HLA-A*02:01:120, HLA-A*02:01:121, HLA-A*02:01:122, HLA-A*02:01:123, HLA-A*02:01:124, HLA-A*02:01:125, HLA-A*02:01:126, HLA-A*02:01:127, HLA-A*02:01:128, HLA-A*02:01:129, HLA-A*02:01:13, HLA-A*02:01:130, HLA-A*02:01:131, HLA-A*02:01:132, HLA-A*02:01:133, HLA-A*02:01:134, HLA-A*02:01:135, HLA-A*02:01:136, HLA-A*02:01:137, HLA-A*02:01:138, HLA-A*02:01:139, HLA-A*02:01:140, HLA-A*02:01:141, HLA-A*02:01:142, HLA-A*02:01:143, HLA-A*02:01:144, HLA-A*02:01:145, HLA-A*02:01:146, HLA-A*02:01:147, HLA-A*02:01:148, HLA-A*02:01:149,HLA-A*02:01:14Q, HLA-A*02:01:15, HLA-A*02:01:150, HLA-A*02:01:151, HLA-A*02:01:152, HLA-A*02:01:153, HLA-A*02:01:154, HLA-A*02:01:155, HLA-A*02:01:156, HLA-A*02:01:157, HLA-A*02:01:158, HLA-A*02:01:159, HLA-A*02:01:160, HLA-A*02:01:161, HLA-A*02:01:17, HLA-A*02:01:18, HLA-A*02:01:19, HLA-A*02:01:21, HLA-A*02:01:22, HLA-A*02:01:23, HLA-A*02:01:24, HLA-A*02:01:25, HLA-A*02:01:26, HLA-A*02:01:27, HLA-A*02:01:28, HLA-A*02:01:29, HLA-A*02:01:30, HLA-A*02:01:31, HLA-A*02:01:32, HLA-A*02:01:33, HLA-A*02:01:34, HLA-A*02:01:35, HLA-A*02:01:36, HLA-A*02:01:37, HLA-A*02:01:38, HLA-A*02:01:39, HLA-A*02:01:40, HLA-A*02:01:41, HLA-A*02:01:42, HLA-A*02:01:43, HLA-A*02:01:44, HLA-A*02:01:45, HLA-A*02:01:46, HLA-A*02:01:47, HLA-A*02:01:48, HLA-A*02:01:49, HLA-A*02:01:50, HLA-A*02:01:51, HLA-A*02:01:52, HLA-A*02:01:53, HLA-A*02:01:54, HLA-A*02:01:55, HLA-A*02:01:56, HLA-A*02:01:57, HLA-A*02:01:58, HLA-A*02:01:59, HLA-A*02:01:60, HLA-A*02:01:61, HLA-A*02:01:62, HLA-A*02:01:63, HLA-A*02:01:64, HLA-A*02:01:65, HLA-A*02:01:66, HLA-A*02:01:67, HLA-A*02:01:68,HLA-A*02:01:69, HLA-A*02:01:70, HLA-A*02:01:71, HLA-A*02:01:72, HLA-A*02:01:73, HLA-A*02:01:74, HLA-A*02:01:75, HLA-A*02:01:76, HLA-A*02:01:77, HLA-A*02:01:78, HLA-A*02:01:79, HLA-A*02:01:80, HLA-A*02:01:81, HLA-A*02:01:83, HLA-A*02:01:84, HLA-A*02:01:85, HLA-A*02:01:86, HLA-A*02:01:87, HLA-A*02:01:88, HLA-A*02:01:89, HLA-A*02:01:90, HLA-A*02:01:91, HLA-A*02:01:92, HLA-A*02:01:93, HLA-A*02:01:94, HLA-A*02:01:95, HLA-A*02:01:96, HLA-A*02:01:97, HLA-A*02:01:98, HLA-A*02:01:99, HLA-A*02:02:01:01, HLA-A*02:02:01:02, HLA-A*02:02:01:03, HLA-A*02:02:01:04, HLA-A*02:02:02, HLA-A*02:02:03, HLA-A*02:02:04, HLA-A*02:03:01, HLA-A*02:03:02, HLA-A*02:03:03, HLA-A*02:03:04, HLA-A*02:03:05, HLA-A*02:03:06, HLA-A*02:03:07, HLA-A*02:03:08, HLA-A*02:04, HLA-A*02:05:01:01, HLA-A*02:05:01:02, HLA-A*02:05:02, HLA-A*02:05:03, HLA-A*02:05:04, HLA-A*02:05:05, HLA-A*02:05:06, HLA-A*02:05:07, HLA-A*02:05:08, HLA-A*02:05:09, HLA-A*02:06:01:01, HLA-A*02:06:01:02, HLA-A*02:06:01:03, HLA-A*02:06:01:04, HLA-A*02:06:01:05, HLA-A*02:06:01:06, HLA-A*02:06:02, HLA-A*02:06:03,HLA-A*02:06:04, HLA-A*02:06:05, HLA-A*02:06:06, HLA-A*02:06:07, HLA-A*02:06:08, HLA-A*02:06:09, HLA-A*02:06:10, HLA-A*02:06:11, HLA-A*02:06:12, HLA-A*02:06:13, HLA-A*02:06:14, HLA-A*02:06:15, HLA-A*02:06:16, HLA-A*02:06:17, HLA-A*02:06:18, HLA-A*02:06:19, HLA-A*02:06:20, HLA-A*02:06:21, HLA-A*02:06:22, HLA-A*02:06:23, HLA-A*02:06:24, HLA-A*02:06:25, HLA-A*02:06:26, HLA-A*02:06:27, HLA-A*02:07:01, HLA-A*02:07:02, HLA-A*02:07:03, HLA-A*02:07:04, HLA-A*02:07:05, HLA-A*02:07:06, HLA-A*02:07:07, HLA-A*02:07:08, HLA-A*02:07:09, HLA-A*02:07:10, HLA-A*02:07:11, HLA-A*02:07:12, HLA-A*02:08, HLA-A*02:09:01:01, HLA-A*02:09:01:02, HLA-A*02:10, HLA-A*02:101:01, HLA-A*02:101:02, HLA-A*02:102, 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HLA-A*02:154, HLA-A*02:155, HLA-A*02:156, HLA-A*02:157:01, HLA-A*02:157:02, HLA-A*02:158, HLA-A*02:159, HLA-A*02:15N, HLA-A*02:16, HLA-A*02:160, HLA-A*02:161, HLA-A*02:162, HLA-A*02:163, HLA-A*02:164:01, HLA-A*02:164:02, HLA-A*02:165, HLA-A*02:166, HLA-A*02:167, HLA-A*02:168, HLA-A*02:169, HLA-A*02:170, HLA-A*02:171:01, HLA-A*02:171:02, HLA-A*02:172, HLA-A*02:173, HLA-A*02:174, HLA-A*02:175, HLA-A*02:176, HLA-A*02:177, HLA-A*02:178, HLA-A*02:179,HLA-A*02:17:02, HLA-A*02:17:03, HLA-A*02:17:04, HLA-A*02:18, HLA-A*02:180, HLA-A*02:181, HLA-A*02:182, HLA-A*02:183, HLA-A*02:184, HLA-A*02:185, HLA-A*02:186, HLA-A*02:187, HLA-A*02:188, HLA-A*02:189, HLA-A*02:19, HLA-A*02:190, HLA-A*02:191, HLA-A*02:192, HLA-A*02:193, HLA-A*02:194, HLA-A*02:195, HLA-A*02:196, HLA-A*02:197:01, HLA-A*02:197:02, HLA-A*02:198, HLA-A*02:199, HLA-A*02:200, HLA-A*02:201, HLA-A*02:202, HLA-A*02:203, HLA-A*02:204, HLA-A*02:205, HLA-A*02:206, HLA-A*02:207, HLA-A*02:208, HLA-A*02:209, HLA-A*02:20:01, HLA-A*02:20:02, HLA-A*02:21, HLA-A*02:210, HLA-A*02:211:01, HLA-A*02:211:02, HLA-A*02:212, HLA-A*02:213, HLA-A*02:214, HLA-A*02:215, HLA-A*02:216, HLA-A*02:217:01, HLA-A*02:217:02, HLA-A*02:218, HLA-A*02:219, HLA-A*02:220, HLA-A*02:221, HLA-A*02:222N, HLA-A*02:223N, HLA-A*02:224, HLA-A*02:225N, HLA-A*02:226N, HLA-A*02:227N, HLA-A*02:228, HLA-A*02:229, HLA-A*02:22:01:01, HLA-A*02:22:01:02, HLA-A*02:22:02, HLA-A*02:230, HLA-A*02:231, HLA-A*02:232, HLA-A*02:233, HLA-A*02:234, HLA-A*02:235, HLA-A*02:236, HLA-A*02:237, HLA-A*02:238,HLA-A*02:239, HLA-A*02:240, HLA-A*02:241, HLA-A*02:242, HLA-A*02:243:01, HLA-A*02:243:02, HLA-A*02:243:03, HLA-A*02:244, HLA-A*02:245, HLA-A*02:246, HLA-A*02:247, HLA-A*02:248, HLA-A*02:249, HLA-A*02:24:01, HLA-A*02:24:02, HLA-A*02:25, HLA-A*02:250N, HLA-A*02:251, HLA-A*02:252, HLA-A*02:253, HLA-A*02:254, HLA-A*02:255, HLA-A*02:256, HLA-A*02:257, HLA-A*02:258, HLA-A*02:259, HLA-A*02:26, HLA-A*02:260, HLA-A*02:261, HLA-A*02:262, HLA-A*02:263, HLA-A*02:264, HLA-A*02:265, 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HLA-A*02:390, HLA-A*02:391, HLA-A*02:392, HLA-A*02:393, HLA-A*02:394, HLA-A*02:395N, HLA-A*02:396, HLA-A*02:397, HLA-A*02:398, HLA-A*02:399, HLA-A*02:400, HLA-A*02:401, HLA-A*02:402, HLA-A*02:403, HLA-A*02:404, HLA-A*02:405, HLA-A*02:406, HLA-A*02:407, HLA-A*02:408, HLA-A*02:409, HLA-A*02:40:01, HLA-A*02:40:02, HLA-A*02:41, HLA-A*02:410, HLA-A*02:411, HLA-A*02:412, HLA-A*02:413, HLA-A*02:414, HLA-A*02:415, HLA-A*02:416, HLA-A*02:417, HLA-A*02:418, HLA-A*02:419:01, HLA-A*02:419:02, HLA-A*02:42, HLA-A*02:420, HLA-A*02:421, HLA-A*02:422, HLA-A*02:423, HLA-A*02:424, HLA-A*02:425, HLA-A*02:426, HLA-A*02:427, HLA-A*02:428, HLA-A*02:429, HLA-A*02:430, HLA-A*02:431, HLA-A*02:432, HLA-A*02:433, HLA-A*02:434, HLA-A*02:435, HLA-A*02:436, HLA-A*02:437,HLA-A*02:438, HLA-A*02:439N, HLA-A*02:43N, HLA-A*02:44, HLA-A*02:440Q, HLA-A*02:441, HLA-A*02:442, HLA-A*02:443, HLA-A*02:444, HLA-A*02:445, HLA-A*02:446, HLA-A*02:447, HLA-A*02:448, HLA-A*02:449, HLA-A*02:45, HLA-A*02:450, HLA-A*02:451, 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HLA-A*02:516N, HLA-A*02:517, HLA-A*02:518, HLA-A*02:519, HLA-A*02:52, HLA-A*02:520, HLA-A*02:521, HLA-A*02:522, HLA-A*02:523, HLA-A*02:524:01, HLA-A*02:524:02, HLA-A*02:525N, HLA-A*02:526, HLA-A*02:527, HLA-A*02:528:01, HLA-A*02:528:02, HLA-A*02:529, HLA-A*02:530, HLA-A*02:531, HLA-A*02:532, HLA-A*02:533, HLA-A*02:534, HLA-A*02:535, HLA-A*02:536, HLA-A*02:537, HLA-A*02:538, HLA-A*02:539, HLA-A*02:53N, HLA-A*02:54, HLA-A*02:540N, HLA-A*02:541, HLA-A*02:542, HLA-A*02:543, HLA-A*02:544, HLA-A*02:545, HLA-A*02:546, HLA-A*02:547, HLA-A*02:548, HLA-A*02:549, HLA-A*02:55, HLA-A*02:550, HLA-A*02:551, HLA-A*02:552, HLA-A*02:553, HLA-A*02:554, HLA-A*02:555, HLA-A*02:556, HLA-A*02:557, HLA-A*02:558, HLA-A*02:559, HLA-A*02:560, HLA-A*02:561, HLA-A*02:562, HLA-A*02:563, HLA-A*02:564, HLA-A*02:565, HLA-A*02:566, HLA-A*02:567, HLA-A*02:568, HLA-A*02:569, HLA-A*02:56:01, HLA-A*02:56:02, HLA-A*02:57,HLA-A*02:570:01, HLA-A*02:570:02, HLA-A*02:571, HLA-A*02:572, HLA-A*02:573, HLA-A*02:574, HLA-A*02:575, 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HLA-A*02:637, HLA-A*02:638, HLA-A*02:639, HLA-A*02:640, HLA-A*02:641, HLA-A*02:642, HLA-A*02:643N, HLA-A*02:644, HLA-A*02:645, HLA-A*02:646, HLA-A*02:647, HLA-A*02:648, HLA-A*02:649, HLA-A*02:64:01, HLA-A*02:64:02, HLA-A*02:65, HLA-A*02:650, HLA-A*02:651, HLA-A*02:652, HLA-A*02:653, HLA-A*02:654, HLA-A*02:655, HLA-A*02:656, HLA-A*02:657, HLA-A*02:658, HLA-A*02:659, HLA-A*02:66, HLA-A*02:660, HLA-A*02:661, HLA-A*02:662, HLA-A*02:663, HLA-A*02:664, HLA-A*02:665, HLA-A*02:666, HLA-A*02:667, HLA-A*02:668, HLA-A*02:669, HLA-A*02:67, HLA-A*02:670, HLA-A*02:671, HLA-A*02:672Q, HLA-A*02:673, HLA-A*02:674, HLA-A*02:675N, HLA-A*02:676, HLA-A*02:677, HLA-A*02:678, HLA-A*02:679, HLA-A*02:68, HLA-A*02:680, HLA-A*02:681, HLA-A*02:682, HLA-A*02:683, HLA-A*02:684, HLA-A*02:685, HLA-A*02:686, HLA-A*02:687, HLA-A*02:688, HLA-A*02:689, HLA-A*02:69, HLA-A*02:690, HLA-A*02:691N, HLA-A*02:692, HLA-A*02:693, HLA-A*02:694, HLA-A*02:695, HLA-A*02:696N, HLA-A*02:697, HLA-A*02:698, HLA-A*02:699, HLA-A*02:70, HLA-A*02:700, HLA-A*02:701, HLA-A*02:702,HLA-A*02:703, HLA-A*02:704, HLA-A*02:705, HLA-A*02:706, HLA-A*02:707, HLA-A*02:708, HLA-A*02:709, HLA-A*02:71, HLA-A*02:710N, HLA-A*02:711, HLA-A*02:712, HLA-A*02:713, HLA-A*02:714, HLA-A*02:715N, HLA-A*02:716, HLA-A*02:717, HLA-A*02:718, HLA-A*02:719, HLA-A*02:72, HLA-A*02:720, HLA-A*02:721, HLA-A*02:722, HLA-A*02:723, HLA-A*02:724, HLA-A*02:725, HLA-A*02:726, HLA-A*02:727, HLA-A*02:728, HLA-A*02:729, HLA-A*02:73, HLA-A*02:730, HLA-A*02:731, HLA-A*02:732, HLA-A*02:733, HLA-A*02:734, HLA-A*02:735, HLA-A*02:736, HLA-A*02:737, HLA-A*02:738, HLA-A*02:739, HLA-A*02:740, HLA-A*02:741, HLA-A*02:742, HLA-A*02:743, HLA-A*02:744, HLA-A*02:745, HLA-A*02:746, HLA-A*02:747, HLA-A*02:748N, HLA-A*02:749, HLA-A*02:74:01, HLA-A*02:74:02, HLA-A*02:75, HLA-A*02:750, HLA-A*02:751, HLA-A*02:752, HLA-A*02:753, HLA-A*02:754, HLA-A*02:755, HLA-A*02:756, HLA-A*02:757, HLA-A*02:758, HLA-A*02:759, HLA-A*02:760N, HLA-A*02:761, HLA-A*02:762, HLA-A*02:763, HLA-A*02:764, HLA-A*02:765, HLA-A*02:766, HLA-A*02:767, HLA-A*02:768, HLA-A*02:769, HLA-A*02:76:01, HLA-A*02:76:02, HLA-A*02:77,HLA-A*02:770, HLA-A*02:771, HLA-A*02:772, HLA-A*02:773N, HLA-A*02:774, HLA-A*02:775N, HLA-A*02:776, HLA-A*02:777, HLA-A*02:778, HLA-A*02:779, HLA-A*02:78, HLA-A*02:780, HLA-A*02:781, HLA-A*02:782, HLA-A*02:783, HLA-A*02:784, HLA-A*02:785, HLA-A*02:786, HLA-A*02:787, HLA-A*02:788N, HLA-A*02:789N, HLA-A*02:790, HLA-A*02:791N, HLA-A*02:792N, HLA-A*02:793N, HLA-A*02:794, HLA-A*02:795, HLA-A*02:796N, HLA-A*02:797N, HLA-A*02:798, HLA-A*02:799, HLA-A*02:79:01, HLA-A*02:79:02, HLA-A*02:80, HLA-A*02:800, HLA-A*02:801, HLA-A*02:802, HLA-A*02:803N, HLA-A*02:804, HLA-A*02:805Q, HLA-A*02:806N, HLA-A*02:807N, HLA-A*02:808, HLA-A*02:809, HLA-A*02:81, HLA-A*02:810, HLA-A*02:811, HLA-A*02:812, HLA-A*02:813, HLA-A*02:814, HLA-A*02:815, HLA-A*02:816, HLA-A*02:817, HLA-A*02:818, HLA-A*02:819, HLA-A*02:820, HLA-A*02:821, HLA-A*02:822, HLA-A*02:823, HLA-A*02:824, HLA-A*02:825, HLA-A*02:82N, HLA-A*02:83N, HLA-A*02:84, HLA-A*02:85, HLA-A*02:86:01, HLA-A*02:86:02, HLA-A*02:87, HLA-A*02:88N, HLA-A*02:89:01, HLA-A*02:89:02, HLA-A*02:90, HLA-A*02:91, HLA-A*02:92, HLA-A*02:93:01,HLA-A*02:93:02, HLA-A*02:94N, HLA-A*02:95, HLA-A*02:96, HLA-A*02:97:01, HLA-A*02:97:02, and HLA-A*02:99.

[0305] II.D.2. HLA-B alleles and complexes thereof

[0306] Certain aspects of the present disclosure relate to a complex comprising an HLA Class I molecule and an epitope, wherein the HLA Class I molecule is an HLA-B allele, and wherein the epitope is a MAGE-A1 epitope disclosed herein. In certain embodiments, the MAGE-A1 epitope comprises, consists of, or consists essentially of SEQ ID NO: 52.

[0307] Other aspects of the present disclosure relate to a complex comprising an HLA Class I molecule and an epitope, wherein the HLA Class I molecule is an HLA-B allele, and wherein the epitope is a MART1 epitope disclosed herein. In certain embodiments, the MART1 epitope comprises, consists of, or consists essentially of SEQ ID NO: 53.

[0308] Other aspects of the present disclosure relate to a complex comprising an HLA Class I molecule and an epitope, wherein the HLA Class I molecule is an HLA-B allele, and wherein the epitope is a MAGE-A3 epitope disclosed herein. In certain embodiments, the MAGE-A3 epitope comprises, consists of, or consists essentially of SEQ ID NO: 54.

[0309] In some embodiments, the HLA-B allele is selected from the group consisting of HLA-B*07, HLA-B*08, HLA-B*13, HLA-B*14, HLA-B*15, HLA-B*18, HLA-B*27, HLA-B*35, HLA-B*37, HLA-B*38, HLA-B*39, HLA-B*40, HLA-B*41, HLA-B*42, HLA-B*44, HLA-B*45, HLA-B*46, HLA-B*47, HLA-B*48, HLA-B*49, HLA-B*50, HLA-B*51, HLA-B*52, HLA-B*53, HLA-B*54, HLA-B*55, HLA-B*56, HLA-B*57, HLA-B*58, HLA-B*59, HLA-B*67, HLA-B*73, HLA-B*78, HLA-B*79, HLA-B*81, HLA-B*82, and HLA-B*83.

[0310] II.D.2.a. HLA-B*07 allele and complexes thereof

[0311] In some embodiments, the HLA-B allele is an HLA-B*07 allele. In certain embodiments, the complex comprises an HLA-B*07 allele and a MAGE-A1 epitope disclosed herein, e.g., an epitope comprising, consisting of, or consisting essentially of SEQ ID NO: 52. In certain embodiments, the HLA-B allele is an HLA-B*07:02 allele. In certain embodiments, the HLA-B allele is an HLA-B*07:03 allele. In certain embodiments, the HLA-B allele is an HLA-B*07:04 allele. In certain embodiments, the HLA-B allele is an HLA-B*07:05 allele. In certain embodiments, the HLA-B allele is an HLA-B*07:06 allele. In particular embodiments, the complex comprises an HLA-B*07:02 allele and a MAGE-A1 epitope comprising, consisting of, or consisting essentially of SEQ ID NO: 52.

[0312] In certain embodiments, the HLA-B allele is selected from the group consisting of HLA-B*07:02:01:01, HLA-B*07:02:01:02, HLA-B*07:02:01:03, HLA-B*07:02:01:04, HLA-B*07:02:01:05, HLA-B*07:02:01:06, HLA-B*07:02:01:07, HLA-B*07:02:01:08, HLA-B*07:02:01:09, HLA-B*07:02:01:10, HLA-B*07:02:01:11, HLA-B*07:02:01:12, HLA-B*07:02:01:13, HLA-B*07:02:01:14, HLA-B*07:02:02, HLA-B*07:02:03, HLA-B*07:02:04, HLA-B*07:02:05, HLA-B*07:02:06, HLA-B*07:02:07, HLA-B*07:02:08, HLA-B*07:02:09, HLA-B*07:02:10, HLA-B*07:02:11, HLA-B*07:02:12, HLA-B*07:02:13, HLA-B*07:02:14, HLA-B*07:02:15, HLA-B*07:02:16, HLA-B*07:02:17, HLA-B*07:02:18, HLA-B*07:02:19, HLA-B*07:02:20, HLA-B*07:02:21, HLA-B*07:02:22, HLA-B*07:02:23, HLA-B*07:02:24, HLA-B*07:02:25, HLA-B*07:02:26, HLA-B*07:02:27, HLA-B*07:02:28, HLA-B*07:02:29, HLA-B*07:02:30, HLA-B*07:02:31, HLA-B*07:02:32, HLA-B*07:02:33, HLA-B*07:02:34, HLA-B*07:02:35, HLA-B*07:02:36, HLA-B*07:02:37, HLA-B*07:02:38, HLA-B*07:02:39, HLA-B*07:02:40, HLA-B*07:02:41, HLA-B*07:02:42, HLA-B*07:02:43, HLA-B*07:02:44, HLA-B*07:02:45, HLA-B*07:02:46, HLA-B*07:02:47, HLA-B*07:02:48, HLA-B*07:02:49,HLA-B*07:02:50, HLA-B*07:02:51, HLA-B*07:02:52, HLA-B*07:02:53, HLA-B*07:02:54, HLA-B*07:02:55, HLA-B*07:02:56, HLA-B*07:02:57, HLA-B*07:02:58, HLA-B*07:02:59, HLA-B*07:02:60, HLA-B*07:02:61, HLA-B*07:02:62, HLA-B*07:02:63, HLA-B*07:02:64, HLA-B*07:02:65, HLA-B*07:02:66, HLA-B*07:02:67, HLA-B*07:02:68, HLA-B*07:02:69, HLA-B*07:02:70, HLA-B*07:02:71, HLA-B*07:02:72, HLA-B*07:02:73, HLA-B*07:03, HLA-B*07:04:01, HLA-B*07:04:02, HLA-B*07:05:01:01, HLA-B*07:05:01:02, HLA-B*07:05:01:03, HLA-B*07:05:01:04, HLA-B*07:05:02, HLA-B*07:05:03, HLA-B*07:05:04, HLA-B*07:05:05, HLA-B*07:05:06, HLA-B*07:05:07, HLA-B*07:05:08, HLA-B*07:05:09, HLA-B*07:06:01, HLA-B*07:06:02, HLA-B*07:06:03, HLA-B*07:07:01, HLA-B*07:07:02, HLA-B*07:08:01, HLA-B*07:08:02, HLA-B*07:09:01, HLA-B*07:09:02, HLA-B*07:10, HLA-B*07:100, HLA-B*07:101, HLA-B*07:102, HLA-B*07:103, HLA-B*07:104, HLA-B*07:105, HLA-B*07:106, HLA-B*07:107, HLA-B*07:108, HLA-B*07:109, HLA-B*07:11, HLA-B*07:110, HLA-B*07:111, HLA-B*07:112, HLA-B*07:113, HLA-B*07:114, HLA-B*07:115, HLA-B*07:116, HLA-B*07:117, HLA-B*07:118,HLA-B*07:119, HLA-B*07:12, HLA-B*07:120, HLA-B*07:121, HLA-B*07:122, HLA-B*07:123, HLA-B*07:124, HLA-B*07:125, HLA-B*07:126, HLA-B*07:127, HLA-B*07:128, HLA-B*07:129, HLA-B*07:13, HLA-B*07:130, HLA-B*07:131, HLA-B*07:132, HLA-B*07:133, HLA-B*07:134, HLA-B*07:135, HLA-B*07:136:01, HLA-B*07:136:02, HLA-B*07:137, HLA-B*07:138, HLA-B*07:139:01, HLA-B*07:139:02, HLA-B*07:14, HLA-B*07:140, HLA-B*07:141, HLA-B*07:142, HLA-B*07:143, HLA-B*07:144, HLA-B*07:145, HLA-B*07:146, HLA-B*07:147, HLA-B*07:148, HLA-B*07:149, HLA-B*07:15, HLA-B*07:150, HLA-B*07:151:01, HLA-B*07:151:02, HLA-B*07:152, HLA-B*07:153, HLA-B*07:154, HLA-B*07:155, HLA-B*07:156, HLA-B*07:157, HLA-B*07:158, HLA-B*07:159, HLA-B*07:16, HLA-B*07:160, HLA-B*07:161, HLA-B*07:162, HLA-B*07:163, HLA-B*07:164, HLA-B*07:165, HLA-B*07:166, HLA-B*07:167, HLA-B*07:168, HLA-B*07:169, HLA-B*07:17, HLA-B*07:170, HLA-B*07:171, HLA-B*07:172, HLA-B*07:173, HLA-B*07:174, HLA-B*07:175, HLA-B*07:176, HLA-B*07:177, HLA-B*07:178, HLA-B*07:179, HLA-B*07:180, HLA-B*07:181, HLA-B*07:182, HLA-B*07:183, HLA-B*07:184, HLA-B*07:185,HLA-B*07:186, HLA-B*07:187, HLA-B*07:188, HLA-B*07:189, HLA-B*07:18:01, HLA-B*07:18:02, HLA-B*07:19, HLA-B*07:190, HLA-B*07:191, HLA-B*07:192, HLA-B*07:193, HLA-B*07:194, HLA-B*07:195, HLA-B*07:196, HLA-B*07:197, HLA-B*07:198, HLA-B*07:199, HLA-B*07:20, HLA-B*07:200, HLA-B*07:201, HLA-B*07:202, HLA-B*07:203, HLA-B*07:204, HLA-B*07:205, HLA-B*07:206, HLA-B*07:207, HLA-B*07:208, HLA-B*07:209, HLA-B*07:21, HLA-B*07:210, HLA-B*07:211, HLA-B*07:212, HLA-B*07:213, HLA-B*07:214, HLA-B*07:215, HLA-B*07:216, HLA-B*07:217, HLA-B*07:218, HLA-B*07:219, HLA-B*07:220, HLA-B*07:221, HLA-B*07:222, HLA-B*07:223, HLA-B*07:224, HLA-B*07:225, HLA-B*07:226, HLA-B*07:227, HLA-B*07:228:01, HLA-B*07:228:02, HLA-B*07:229, HLA-B*07:22:01, HLA-B*07:22:02, HLA-B*07:23, HLA-B*07:230, HLA-B*07:231, HLA-B*07:232, HLA-B*07:233, HLA-B*07:234, HLA-B*07:235, HLA-B*07:236, HLA-B*07:237, HLA-B*07:238, HLA-B*07:239, HLA-B*07:24, HLA-B*07:240, HLA-B*07:241, HLA-B*07:242, HLA-B*07:243, HLA-B*07:244, HLA-B*07:245, HLA-B*07:246, HLA-B*07:247, HLA-B*07:248, HLA-B*07:249, HLA-B*07:25, HLA-B*07:250,HLA-B*07:251, HLA-B*07:252, HLA-B*07:253, HLA-B*07:254, HLA-B*07:255, HLA-B*07:256, HLA-B*07:257, HLA-B*07:258, HLA-B*07:259, HLA-B*07:26, HLA-B*07:260, HLA-B*07:261, HLA-B*07:262, HLA-B*07:263, HLA-B*07:264, HLA-B*07:265, HLA-B*07:266, HLA-B*07:267, HLA-B*07:268, HLA-B*07:269, HLA-B*07:27, HLA-B*07:270, HLA-B*07:271, HLA-B*07:272, HLA-B*07:273, HLA-B*07:274, HLA-B*07:275, HLA-B*07:276:01, HLA-B*07:276:02, HLA-B*07:277, HLA-B*07:278, HLA-B*07:279, HLA-B*07:28, HLA-B*07:280, HLA-B*07:281, HLA-B*07:282, HLA-B*07:283, HLA-B*07:284, HLA-B*07:285, HLA-B*07:286, HLA-B*07:287, HLA-B*07:288, HLA-B*07:289, HLA-B*07:29, HLA-B*07:290, HLA-B*07:291, HLA-B*07:292, HLA-B*07:293, HLA-B*07:294, HLA-B*07:295, HLA-B*07:296, HLA-B*07:297, HLA-B*07:298, HLA-B*07:299, HLA-B*07:30, HLA-B*07:300, HLA-B*07:301, HLA-B*07:302, HLA-B*07:303:01, HLA-B*07:303:02, HLA-B*07:304, HLA-B*07:305, HLA-B*07:306, HLA-B*07:307, HLA-B*07:308, HLA-B*07:309, HLA-B*07:31, HLA-B*07:310, HLA-B*07:311, HLA-B*07:312, HLA-B*07:313, HLA-B*07:314, HLA-B*07:315, HLA-B*07:316, HLA-B*07:317, HLA-B*07:318,HLA-B*07:319, HLA-B*07:32, HLA-B*07:320, HLA-B*07:321, HLA-B*07:322, HLA-B*07:323, HLA-B*07:324, HLA-B*07:325, HLA-B*07:326, HLA-B*07:327, HLA-B*07:328, HLA-B*07:329, HLA-B*07:330, HLA-B*07:331, HLA-B*07:332, HLA-B*07:333, HLA-B*07:334, HLA-B*07:335, HLA-B*07:336, HLA-B*07:337, HLA-B*07:338, HLA-B*07:339, HLA-B*07:33:01, HLA-B*07:33:02, HLA-B*07:33:03, HLA-B*07:34, HLA-B*07:340, HLA-B*07:341, HLA-B*07:342, HLA-B*07:343, HLA-B*07:344, HLA-B*07:345, HLA-B*07:346, HLA-B*07:347, HLA-B*07:348, HLA-B*07:349, HLA-B*07:35, HLA-B*07:350, HLA-B*07:351, HLA-B*07:352, HLA-B*07:353, HLA-B*07:354, HLA-B*07:355, HLA-B*07:356, HLA-B*07:357, HLA-B*07:358, HLA-B*07:36, HLA-B*07:37:01, HLA-B*07:37:02, HLA-B*07:38, HLA-B*07:39, HLA-B*07:40, HLA-B*07:41, HLA-B*07:42, HLA-B*07:43, HLA-B*07:44, HLA-B*07:45, HLA-B*07:46, HLA-B*07:47, HLA-B*07:48, HLA-B*07:49, HLA-B*07:50, HLA-B*07:51, HLA-B*07:52, HLA-B*07:53, HLA-B*07:54, HLA-B*07:55, HLA-B*07:56:01, HLA-B*07:56:02, HLA-B*07:57, HLA-B*07:58, HLA-B*07:59, HLA-B*07:60, HLA-B*07:61, HLA-B*07:62, HLA-B*07:63, HLA-B*07:64, HLA-B*07:65,HLA-B*07:66, HLA-B*07:67, HLA-B*07:68:01, HLA-B*07:68:02, HLA-B*07:68:03, HLA-B*07:69, HLA-B*07:70, HLA-B*07:71, HLA-B*07:72, HLA-B*07:73, HLA-B*07:74, HLA-B*07:75:01:01, HLA-B*07:75:01:02, HLA-B*07:76, HLA-B*07:77, HLA-B*07:78, HLA-B*07:79, HLA-B*07:80, HLA-B*07:81, HLA-B*07:82, HLA-B*07:83, HLA-B*07:84, HLA-B*07:85:01, HLA-B*07:85:02, HLA-B*07:86, HLA-B*07:87, HLA-B*07:88, HLA-B*07:89, HLA-B*07:90, HLA-B*07:91, HLA-B*07:92, HLA-B*07:93, HLA-B*07:94, HLA-B*07:95, HLA-B*07:96:01, HLA-B*07:96:02, HLA-B*07:97, HLA-B*07:98, and HLA-B*07:99.

[0313] II.D.2.b. HLA-B*18 Alleles and Complexes Thereof

[0314] In some embodiments, the HLA-B allele is an HLA-B*18 allele. In certain embodiments, the complex comprises an HLA-B*18 allele and a MART1 epitope disclosed herein, e.g., an epitope comprising, consisting of, or consisting essentially of SEQ ID NO: 53. In certain embodiments, the HLA-B allele is an HLA-B*18:01 allele. In particular embodiments, the complex comprises an HLA-B*18:01 allele and a MART1 epitope comprising, consisting of, or consisting essentially of SEQ ID NO: 53.

[0315] In some embodiments, the HLA-B allele is an HLA-B*18 allele. In certain embodiments, the complex comprises an HLA-B*18 allele and a MAGE-A3 epitope disclosed herein, e.g., an epitope comprising, consisting of, or consisting essentially of SEQ ID NO: 54. In certain embodiments, the HLA-B allele is an HLA-B*18:01 allele. In particular embodiments, the complex comprises an HLA-B*18:01 allele and a MAGE-A3 epitope comprising, consisting of, or consisting essentially of SEQ ID NO: 54.

[0316] In certain embodiments, the HLA-B allele is selected from the group consisting of HLA-B*18:01:01:01, HLA-B*18:01:01:02, HLA-B*18:01:01:03, HLA-B*18:01:01:04, HLA-B*18:01:01:05, HLA-B*18:01:01:06, HLA-B*18:01:01:07, HLA-B*18:01:01:08, HLA-B*18:01:01:09, HLA-B*18:01:01:10, HLA-B*18:01:01:11, HLA-B*18:01:01:12, HLA-B*18:01:01:13, HLA-B*18:01:01:14, HLA-B*18:01:01:15, HLA-B*18:01:01:16, HLA-B*18:01:01:17, HLA-B*18:01:01:18, HLA-B*18:01:01:19, HLA-B*18:01:02, HLA-B*18:01:03, HLA-B*18:01:04, HLA-B*18:01:05, HLA-B*18:01:06, HLA-B*18:01:07, HLA-B*18:01:08, HLA-B*18:01:09, HLA-B*18:01:10, HLA-B*18:01:11, HLA-B*18:01:12, HLA-B*18:01:13, HLA-B*18:01:14, HLA-B*18:01:15, HLA-B*18:01:16, HLA-B*18:01:17, HLA-B*18:01:18, HLA-B*18:01:19, HLA-B*18:01:20, HLA-B*18:01:21, HLA-B*18:01:22, HLA-B*18:01:23, HLA-B*18:01:24, HLA-B*18:01:25, HLA-B*18:01:26, HLA-B*18:01:27, HLA-B*18:01:28, HLA-B*18:01:29, HLA-B*18:01:30, HLA-B*18:01:31, and HLA-B*18:01:32.

[0317] In certain embodiments, the HLA-B allele is selected from the group consisting of HLA-B*18:02, HLA-B*18:03:01, HLA-B*18:03:02, HLA-B*18:04:01, HLA-B*18:04:02, HLA-B*18:05:01:01, HLA-B*18:05:01:02, HLA-B*18:06, HLA-B*18:07:01, HLA-B*18:07:02, HLA-B*18:08, HLA-B*18:09, HLA-B*18:10, HLA-B*18:100, HLA-B*18:101, HLA-B*18:102, HLA-B*18:103, HLA-B*18:104, HLA-B*18:105, HLA-B*18:106, HLA-B*18:107, HLA-B*18:108, HLA-B*18:109, HLA-B*18:11, HLA-B*18:110, HLA-B*18:111, HLA-B*18:112, HLA-B*18:113, HLA-B*18:114, HLA-B*18:115, HLA-B*18:116, HLA-B*18:117, HLA-B*18:118, HLA-B*18:119, HLA-B*18:120, HLA-B*18:121, HLA-B*18:122, HLA-B*18:123, HLA-B*18:124, HLA-B*18:125, HLA-B*18:126, HLA-B*18:127, HLA-B*18:128, HLA-B*18:129, HLA-B*18:12:01, HLA-B*18:12:02, HLA-B*18:13, HLA-B*18:130, HLA-B*18:131:01:01, HLA-B*18:131:01:02, HLA-B*18:132, HLA-B*18:133, HLA-B*18:134, HLA-B*18:135, HLA-B*18:136, HLA-B*18:137, HLA-B*18:138N, HLA-B*18:139, HLA-B*18:14, HLA-B*18:140, HLA-B*18:141, HLA-B*18:142, HLA-B*18:143, HLA-B*18:144, HLA-B*18:145, HLA-B*18:146, HLA-B*18:147, HLA-B*18:148, HLA-B*18:149, HLA-B*18:15, HLA-B*18:150, HLA-B*18:151,HLA-B*18:152, HLA-B*18:153, HLA-B*18:154N, HLA-B*18:155, HLA-B*18:156:01:01, HLA-B*18:156:01:02, HLA-B*18:157:01:01, HLA-B*18:157:01:02, HLA-B*18:158, HLA-B*18:159, HLA-B*18:160, HLA-B*18:161, HLA-B*18:17N, HLA-B*18:18:01:01, HLA-B*18:18:01:02, HLA-B*18:19, HLA-B*18:20, HLA-B*18:21, HLA-B*18:22, HLA-B*18:23N, HLA-B*18:24, HLA-B*18:25, HLA-B*18:26, HLA-B*18:27, HLA-B*18:28, HLA-B*18:29, HLA-B*18:30, HLA-B*18:31, HLA-B*18:32, HLA-B*18:33, HLA-B*18:34, HLA-B*18:35, HLA-B*18:36, HLA-B*18:37:01, HLA-B*18:37:02, HLA-B*18:38, HLA-B*18:39, HLA-B*18:40, HLA-B*18:41, HLA-B*18:42, HLA-B*18:43, HLA-B*18:44:01, HLA-B*18:44:02, HLA-B*18:45, HLA-B*18:46, HLA-B*18:47, HLA-B*18:48, HLA-B*18:49, HLA-B*18:50, HLA-B*18:51, HLA-B*18:52, HLA-B*18:53, HLA-B*18:54, HLA-B*18:55, HLA-B*18:56, HLA-B*18:57:01, HLA-B*18:57:02, HLA-B*18:58, HLA-B*18:59, HLA-B*18:60, HLA-B*18:61, HLA-B*18:62, HLA-B*18:63, HLA-B*18:64, HLA-B*18:65, HLA-B*18:66, HLA-B*18:67, HLA-B*18:68, HLA-B*18:69, HLA-B*18:70, HLA-B*18:71, HLA-B*18:72:01, HLA-B*18:72:02, HLA-B*18:72:03, HLA-B*18:73, HLA-B*18:74N,HLA-B*18:75, HLA-B*18:76, HLA-B*18:77, HLA-B*18:78, HLA-B*18:79, HLA-B*18:80, HLA-B*18:81, HLA-B*18:82, HLA-B*18:83, HLA-B*18:84, HLA-B*18:85, HLA-B*18:86, HLA-B*18:87, HLA-B*18:88, HLA-B*18:89, HLA-B*18:90, HLA-B*18:91, HLA-B*18:92, HLA-B*18:93, HLA-B*18:94N, HLA-B*18:95, HLA-B*18:96, HLA-B*18:97, HLA-B*18:98, and HLA-B*18:99.

[0318] II.D.3. HLA-C alleles and complexes thereof

[0319] Certain aspects of the present disclosure relate to a complex comprising an HLA Class I molecule and an epitope, wherein the HLA Class I molecule is an HLA-C allele, and wherein the epitope is a tyrosinase epitope disclosed herein. In certain embodiments, the tyrosinase epitope comprises, consists of, or consists essentially of SEQ ID NO: 51.

[0320] In some embodiments, the HLA-C allele is selected from the group consisting of an HLA-C*05:01 allele, an HLA-C*05:03 allele, an HLA-C*05:04 allele, an HLA-C*05:05 allele, and an HLA-C*05:06 allele. In certain embodiments, the HLA-C allele is an HLA-C*05:01 allele. In certain embodiments, the HLA-C allele is an HLA-C*05:03 allele. In certain embodiments, the HLA-C allele is an HLA-C*05:04 allele. In certain embodiments, the HLA-C allele is an HLA-C*05:05 allele. In certain embodiments, the HLA-C allele is an HLA-C*05:06 allele.

[0321] In certain embodiments, the complex comprises an HLA-C*05 allele and a tyrosinase epitope disclosed herein, e.g., an epitope comprising, consisting of, or consisting essentially of SEQ ID NO: 51. In certain embodiments, the HLA-C allele is an HLA-C*05:01 allele. In particular embodiments, the complex comprises an HLA-C*05:01 allele and a tyrosinase epitope comprising, consisting of, or consisting essentially of SEQ ID NO: 51.

[0322] In certain embodiments, the HLA-C allele is selected from the group consisting of HLA-C*05:01:01:01, HLA-C*05:01:01:02, HLA-C*05:01:01:03, HLA-C*05:01:01:04, HLA-C*05:01:01:05, HLA-C*05:01:01:06, HLA-C*05:01:01:07, HLA-C*05:01:01:08, HLA-C*05:01:01:09, HLA-C*05:01:01:10, HLA-C*05:01:01:11, HLA-C*05:01:01:12, HLA-C*05:01:01:13, HLA-C*05:01:01:14, HLA-C*05:01:01:15, HLA-C*05:01:01:16, HLA-C*05:01:02, HLA-C*05:01:03, HLA-C*05:01:04, HLA-C*05:01:05, HLA-C*05:01:06, HLA-C*05:01:07, HLA-C*05:01:08, HLA-C*05:01:09, HLA-C*05:01:10, HLA-C*05:01:11, HLA-C*05:01:12, HLA-C*05:01:13, HLA-C*05:01:14, HLA-C*05:01:15, HLA-C*05:01:16, HLA-C*05:01:17, HLA-C*05:01:18, HLA-C*05:01:19, HLA-C*05:01:20, HLA-C*05:01:21, HLA-C*05:01:22, HLA-C*05:01:23, HLA-C*05:01:24, HLA-C*05:01:25, HLA-C*05:01:26, HLA-C*05:01:27, HLA-C*05:01:28, HLA-C*05:01:29, HLA-C*05:01:30, HLA-C*05:01:31, HLA-C*05:01:32, HLA-C*05:01:33, HLA-C*05:01:34, HLA-C*05:01:35, HLA-C*05:01:36, HLA-C*05:01:37, HLA-C*05:01:38, HLA-C*05:01:39, HLA-C*05:01:40, HLA-C*05:01:41, HLA-C*05:01:42, HLA-C*05:01:43, HLA-C*05:01:44, HLA-C*05:01:45, HLA-C*05:03,HLA-C*05:04:01, HLA-C*05:04:02, HLA-C*05:05:01, HLA-C*05:05:02, HLA-C*05:06, HLA-C*05:07N, HLA-C*05:08, HLA-C*05:09:01, HLA-C*05:09:02, HLA-C*05:09:03, HLA-C*05:10, HLA-C*05:100, HLA-C*05:101, HLA-C*05:102, HLA-C*05:103:01, HLA-C*05:103:02, HLA-C*05:104, HLA-C*05:105, HLA-C*05:106:01, HLA-C*05:106:02, HLA-C*05:107, HLA-C*05:108, HLA-C*05:109, HLA-C*05:11, HLA-C*05:110, HLA-C*05:111, HLA-C*05:112, HLA-C*05:113N, HLA-C*05:114, HLA-C*05:115, HLA-C*05:116, HLA-C*05:117, HLA-C*05:118, HLA-C*05:119, HLA-C*05:12, HLA-C*05:120, HLA-C*05:121, HLA-C*05:122, HLA-C*05:123, HLA-C*05:124, HLA-C*05:125, HLA-C*05:126, HLA-C*05:127, HLA-C*05:128N, HLA-C*05:129, HLA-C*05:13, HLA-C*05:130, HLA-C*05:131, HLA-C*05:132, HLA-C*05:133, HLA-C*05:134, HLA-C*05:135, HLA-C*05:136, HLA-C*05:137, HLA-C*05:138, HLA-C*05:139, HLA-C*05:14, HLA-C*05:140, HLA-C*05:141, HLA-C*05:142, HLA-C*05:143, HLA-C*05:144, HLA-C*05:145, HLA-C*05:146, HLA-C*05:147, HLA-C*05:148, HLA-C*05:149, HLA-C*05:15, HLA-C*05:150, HLA-C*05:151, HLA-C*05:152, HLA-C*05:153N, HLA-C*05:154N, HLA-C*05:155, HLA-C*05:156,HLA-C*05:157, HLA-C*05:158, HLA-C*05:159, HLA-C*05:16, HLA-C*05:160, HLA-C*05:161, HLA-C*05:162, HLA-C*05:163, HLA-C*05:164, HLA-C*05:165, HLA-C*05:166, HLA-C*05:167, HLA-C*05:168, HLA-C*05:169N, HLA-C*05:17, HLA-C*05:170, HLA-C*05:171:01:01, HLA-C*05:171:01:02, HLA-C*05:172, HLA-C*05:173, HLA-C*05:174, HLA-C*05:175N, HLA-C*05:176, HLA-C*05:177, HLA-C*05:178, HLA-C*05:179, HLA-C*05:180N, HLA-C*05:181, HLA-C*05:182, HLA-C*05:183, HLA-C*05:184, HLA-C*05:185, HLA-C*05:186, HLA-C*05:187, HLA-C*05:188, HLA-C*05:189, HLA-C*05:18:01, HLA-C*05:18:02, HLA-C*05:18:03, HLA-C*05:18:04, HLA-C*05:18:05, HLA-C*05:19, HLA-C*05:190, HLA-C*05:191, HLA-C*05:192, HLA-C*05:193, HLA-C*05:194, HLA-C*05:195, HLA-C*05:196, HLA-C*05:197, HLA-C*05:198, HLA-C*05:199, HLA-C*05:20, HLA-C*05:200, HLA-C*05:201, HLA-C*05:202Q, HLA-C*05:203, HLA-C*05:21, HLA-C*05:22:01, HLA-C*05:22:02, HLA-C*05:23, HLA-C*05:24, HLA-C*05:25, HLA-C*05:26, HLA-C*05:27, HLA-C*05:28, HLA-C*05:29:01, HLA-C*05:29:02, HLA-C*05:30, HLA-C*05:31, HLA-C*05:32, HLA-C*05:33, HLA-C*05:34, HLA-C*05:35, HLA-C*05:36,HLA-C*05:37, HLA-C*05:38, HLA-C*05:39, HLA-C*05:40, HLA-C*05:41, HLA-C*05:42, HLA-C*05:43, HLA-C*05:44:01, HLA-C*05:44:02, HLA-C*05:45, HLA-C*05:46, HLA-C*05:47, HLA-C*05:48N, HLA-C*05:49, HLA-C*05:50, HLA-C*05:51Q, HLA-C*05:52, HLA-C*05:53, HLA-C*05:54, HLA-C*05:55, HLA-C*05:56, HLA-C*05:57, HLA-C*05:58:01, HLA-C*05:58:02, HLA-C*05:58:03, HLA-C*05:58:04, HLA-C*05:59, HLA-C*05:60, HLA-C*05:61, HLA-C*05:62, HLA-C*05:63, HLA-C*05:64:01, HLA-C*05:64:02, HLA-C*05:65, HLA-C*05:66, HLA-C*05:67, HLA-C*05:68, HLA-C*05:69, HLA-C*05:70, HLA-C*05:71, HLA-C*05:72, HLA-C*05:73, HLA-C*05:74, HLA-C*05:75, HLA-C*05:76, HLA-C*05:77, HLA-C*05:78:01, HLA-C*05:78:02, HLA-C*05:79, HLA-C*05:80, HLA-C*05:81, HLA-C*05:82, HLA-C*05:83, HLA-C*05:84, HLA-C*05:85, HLA-C*05:86, HLA-C*05:87, HLA-C*05:88, HLA-C*05:89, HLA-C*05:90, HLA-C*05:91N, HLA-C*05:92N, HLA-C*05:93, HLA-C*05:94, HLA-C*05:95, HLA-C*05:96, HLA-C*05:97, HLA-C*05:98, and HLA-C*05:99N.

[0323] II.E. TCR-expressing cells

[0324] Certain aspects of the present disclosure relate to a cell comprising a nucleic acid molecule disclosed herein, a vector disclosed herein, a recombinant TCR disclosed herein, a bispecific TCR disclosed herein, or any combination thereof. Any cell can be used in the present disclosure.

[0325] In certain embodiments, the cell expresses CD3. The CD3 expression can be naturally occurring, e.g., CD3 is expressed from a nucleic acid sequence endogenously expressed by the cell. For example, T cells and natural killer (NK) cells naturally express CD3. Thus, in some embodiments, the cell is a T cell or a natural killer cell. In certain embodiments, the cell is a T cell selected from a natural killer T (NKT) cell and an innate lymphoid cell (ILC).

[0326] In some embodiments, the T cell is isolated from a human subject. In some embodiments, the human subject is the same subject who will ultimately receive the T cell therapy. In other embodiments, the subject is a donor subject, wherein the donor subject is not the same subject who will receive the T cell therapy.

[0327] In some embodiments, the cell is a cell that does not naturally express CD3, wherein the cell is modified to express CD3. In some embodiments, the cell comprises a transgene encoding CD3, wherein the transgene is expressed by the cell. In some embodiments, the cell comprises a transgene encoding a protein that activates the cell to express an endogenous CD3. In some embodiments, the cell comprises a transgene encoding a protein or siRNA that inhibits a CD3 expression inhibitor in the cell. In some embodiments, the transgene is incorporated into the genome of the cell. In some embodiments, the transgene is not incorporated into the genome of the cell.

[0328] In some embodiments, the cell modified to express CD3 is isolated from a human subject. In some embodiments, the human subject is the same subject who will ultimately receive the cell therapy. In other embodiments, the subject is a donor subject, wherein the donor subject is not the same subject who will receive the cell therapy.

[0329] II. F. Vaccines

[0330] Certain aspects of the present disclosure are a cancer vaccine comprising a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, the cancer vaccine comprises a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the vaccine further comprises one or more excipients. In some embodiments, the vaccine further comprises one or more additional peptides. In some embodiments, the one or more additional peptides comprise one or more additional epitopes.

[0331] III. Methods of the Present Disclosure

[0332] Certain aspects of the present disclosure relate to methods of treating cancer in a subject in need thereof. Other aspects of the present disclosure relate to methods of engineering antigen-targeting cells. Other aspects of the present disclosure relate to methods of enriching a target T cell population obtained from a human subject.

[0333] III.A. Methods of treating cancer

[0334] Certain aspects of the present disclosure relate to methods of treating cancer in a subject in need thereof, comprising administering to the subject a nucleic acid molecule disclosed herein, a recombinant TCR disclosed herein, a bispecific TCR disclosed herein, an epitope disclosed herein, or an HLA Class I molecule disclosed herein, or a vector or cell comprising any of the foregoing.

[0335] In some embodiments, the cancer is selected from melanoma, bone cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, cutaneous or intraocular malignant melanoma, pancreatic cancer, skin cancer, cancer of the head or neck, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of said cancers. In some embodiments, the cancer is melanoma.

[0336] In some embodiments, the cancer is a relapsed cancer. In some embodiments, the cancer is a refractory cancer. In some embodiments, the cancer is an advanced cancer. In some embodiments, the cancer is a metastatic cancer.

[0337] In some embodiments, the methods disclosed herein treat a cancer in a subject. In some embodiments, the methods disclosed herein reduce the severity of one or more symptoms of a cancer. In some embodiments, the methods disclosed herein reduce the size or number of tumors derived from a cancer. In some embodiments, the methods disclosed herein increase the overall survival of a subject relative to a subject not provided the methods disclosed herein. In some embodiments, the methods disclosed herein increase the progression-free survival of a subject relative to a subject not provided the methods disclosed herein. In some embodiments, the methods disclosed herein produce a partial response in a subject. In some embodiments, the methods disclosed herein produce a complete response in a subject.

[0338] In some embodiments, the methods disclosed herein comprise treating a cancer in a subject in need thereof comprising administering to the subject a cell described herein, wherein the cell comprises a nucleic acid molecule disclosed herein, a vector disclosed herein, a recombinant TCR disclosed herein, and / or a bispecific antibody disclosed herein. In some embodiments, the cell is a T cell. In some embodiments, the cell is a cell modified to express CD3.

[0339] In some embodiments, the cell, e.g., T cell, is obtained from the subject. In some embodiments, the cell, e.g., T cell, is obtained from a donor other than the subject.

[0340] In some embodiments, the subject is pre-conditioned prior to administration of the cell. Pre-conditioning can comprise any agent that promotes T cell function and / or survival. In some embodiments, pre-conditioning comprises administering to the subject a chemotherapy, a cytokine, a protein, a small molecule, or any combination thereof. In some embodiments, pre-conditioning comprises administering an interleukin. In some embodiments, pre-conditioning comprises administering IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof. In some embodiments, pre-conditioning comprises administering cyclophosphamide, fludarabine, or both. In some embodiments, pre-conditioning comprises administering vitamin C, an AKT inhibitor, ATRA (vesanoid, tretinoin), rapamycin, or any combination thereof.

[0341] III.B. Methods of engineering antigen-targeting cells

[0342] Certain aspects of the present disclosure relate to methods of engineering antigen targeting cells. In some embodiments, the antigen is selected from the group consisting of a tyrosinase antigen, a MAGE-A1 antigen, a MART1 antigen, a MAGE-A3 antigen, a SSX antigen, and any combination thereof. In some embodiments, the method comprises transducing a cell with a nucleic acid molecule disclosed herein or a vector disclosed herein. The cell can be any cell described herein. In some embodiments, the cell is a T cell described herein. In some embodiments, the cell is a cell modified to express CD3 as described herein. In some embodiments, the cell, e.g., T cell, is obtained from a subject in need of a T cell therapy. In some embodiments, the cell is obtained from a donor other than a subject in need of a T cell therapy. In some embodiments, the cell is a T cell or a natural killer cell.

[0343] III.C. Methods of enriching a target T cell population

[0344] Certain aspects of the present disclosure relate to methods of enriching a target T cell population obtained from a human subject. In some embodiments, the method comprises contacting a T cell with an HLA class I molecule disclosed herein. In some embodiments, the method comprises contacting a T cell with an APC disclosed herein. In some embodiments, the enriched T cell population comprises a higher number of T cells capable of binding to an HLA class I molecule after the contacting relative to the number of T cells capable of binding to an HLA class I molecule prior to the contacting.

[0345] In some embodiments, the method comprises contacting a T cell in vitro with a peptide, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 51, 52, 53, 54, 55, and any combination thereof. In some embodiments, the method comprises contacting a T cell in vitro with a peptide, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 51, 52, 53, 54, 55, and any combination thereof. In some embodiments, the enriched T cell population comprises a higher number of T cells capable of binding to an HLA class I molecule after the contacting relative to the number of T cells capable of binding to an HLA class I molecule prior to the contacting.

[0346] Some aspects of the present disclosure relate to a method of selecting a T cell capable of targeting a tumor cell. In some embodiments, the method comprises contacting an isolated T cell population in vitro with a peptide, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 51, 52, 53, 54, 55, and any combination thereof. In some embodiments, the T cell is obtained from a human subject.

[0347] T cells obtained from a human subject can be any T cell disclosed herein. In some embodiments, the T cells obtained from a human subject are tumor infiltrating lymphocytes (TILs).

[0348] In some embodiments, the method further comprises administering the enriched T cells to the human subject. In some embodiments, the subject is pre-conditioned prior to receiving the T cells, as described herein.

[0349] All of the various aspects, embodiments, and alternatives described herein can be combined in any and all variations.

[0350] 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.

[0351] Having generally described this disclosure, a further understanding can be obtained by reference to the examples provided herein. These examples are for the purpose of illustration only and are not intended to be limiting.

[0352] Examples

[0353] Example 1 - Methods

[0354] Cell samples

[0355] Peripheral blood samples were obtained from healthy donors. Mononuclear cells were obtained via density gradient centrifugation (Ficoll-Paque PLUS; GE Healthcare). K562 is a red-white blood leukemia cell line with defective HLA expression. T2 is an HLA-A*02:01 + T cell leukemia / B-LCL hybrid cell lines. Jurkat 76 is a T cell leukemia cell line lacking TCR and CD8 expression. K562, T2, and Jurkat 76 cell lines were cultured in RPMI1640 supplemented with 10% FBS and 50 pg / ml gentamycin (Invitrogen). TILs isolated from a metastatic melanoma patient were grown in vitro.

[0356] Tyrosinase: Me275 and MCF7 cell lines were grown in DMEM supplemented with 10% FBS and 50 pg / ml gentamycin (Invitrogen). Melme-3M cell line was grown in IMDM supplemented with 20% FBS and 50 pg / ml gentamycin (Invitrogen).

[0357] MAGE-A1 : Me275, SK-MEL-37 and SK-MEL-21 cell lines were grown in DMEM supplemented with 10% FBS and 50 pg / ml gentamycin (Invitrogen).

[0358] MART1 : A375 and SK-MEL-28 cell lines were grown in DMEM supplemented with 10% FBS and 50 pg / ml gentamycin (Invitrogen). Malme-3M cell line was grown in IMDM supplemented with 20% FBS and 50 pg / ml gentamycin.

[0359] MAGE-A3: SK-MEL-28 and HEK293T cell lines were grown in DMEM supplemented with 10% FBS and 50 pg / ml gentamycin (Invitrogen).

[0360] SSX2: SK-MEL-21, SK-MEL-37 and SK-MEL-28 cell lines were grown in DMEM supplemented with 10% FBS and 50 pg / ml gentamycin (Invitrogen).

[0361] Peptides

[0362] Synthetic peptides were dissolved in DMSO to 50 pg / ml.

[0363] Tyrosinase: The peptide used was C*05:01 restricted tyrosinase 460-468 (FQDYIKSYL; SEQ ID NO: 51) and HIV rev 67-75 (SAEPVPLQL; SEQ ID NO: 82) peptides. The HIV rev 67-75 peptide was used as a negative control.

[0364] MAGE-A1 : The peptide used was B*07:02 restricted MAGE-A1 289-297 (RVRFFFPSL; SEQ ID NO: 52), NY-ESO-1 60-72 (APRGPHGGAASGL; SEQ ID NO: 83), EBV EBNA3A 379-387 (RPPIFIRRL; SEQ ID NO: 84) and HIV nef 128-137 (TPGPGVRYPL; SEQ ID NO: 85) peptides. The NY-ESO-1 60-72 , EBV EBNA3A 379-387 and HIV nef 128-137 peptides were used as negative controls.

[0365] MART1 : The peptides used were 20-mer overlapping peptides covering the entire protein of MART1 and B*18:01 restricted MART1 25-33 (MEVDPIGHLY; SEQ ID NO: 54), MART1 167-176 (EEAAGIGIL; SEQ ID NO: 53) and HIV gag 161-170 (FRDYVDRFYK; SEQ ID NO: 86) peptides. MART1 167-176 and HIV gag 161-170 peptides were used as negative controls.

[0366] MART1 : The peptides used were 20-mer overlapping peptides covering the entire protein of MART1 and B*18:01 restricted MART1 167-176 (MEVDPIGHLY; SEQ ID NO: 54), MART1 25-33 (EEAAGIGIL; SEQ ID NO: 53) and HIV gag 161-170 (FRDYVDRFYK; SEQ ID NO: 86) peptides. MART1 25-33 and HIV gag 161-170 peptides were used as negative controls.

[0367] SSX2: The peptides used were A*02:01 restricted SSX2 41-49 (KASEKIFYV; SEQ ID NO: 55), NY-ESO-1 157-165 (SLLMWITQV; SEQ ID NO: 87) and HTLV-1 tax 11-19 (LLFGYPVYV; SEQ ID NO: 88) peptides. NY-ESO-1 157-165 and HTLV-1 tax 11-19 peptides were used as negative controls.

[0368] gene

[0369] Where applicable, each of the HLA C*05:01, B*07:02, and B*18:01 genes were fused via an internal ribosome entry site to a truncated version of human nerve growth factor receptor (ΔNGFR). ΔNGFR transduced cells were isolated using an anti-NGFR mAb. The full-length tyrosinase gene was cloned from SK-MEL-28 cells via RT-PCR according to published sequences. The full-length MART1 gene was cloned from Malme-3M cells via RT-PCR according to published sequences. The full-length SSX2 gene was cloned from SK-MEL-37 cells via RT-PCR according to published sequences. TCR genes were cloned by 5'-rapid amplification of cDNA ends (RACE) PCR of cDNA ends using the SMARTer RACE cDNA Amplification Kit (Takara Bio). The 5'-RACE PCR products were cloned into retroviral vectors and sequenced. All genes were cloned into pMX retroviral vectors and transduced using a 293GPG cell-based retroviral system.

[0370] Transfectants

[0371] Jurkat 76 / CD8 cells were transduced with individual TCRa and TCRP genes. Jurkat 76 / CD8 derived TCR transfectants were purified (>95% purity) using CD3 microbeads (Miltenyi Biotec). K562-based artificial APCs expressing individual HLA class I genes as single HLA alleles in combination with CD80 and CD83 have been previously reported (Butler and Hirano, Immunol. Rev. 257: 191-209 (2014); Hirano et al., Clin. Cancer Res. 12: 2967-75 (2006)). TCR genes were transduced into human primary T cells using PG13-derived retroviral supernatants. TCR genes were transfected into the 293GPG cell line using TransIT293 (Mirus Bio).

[0372] Tyrosinase - MCF7 cells to generate MCF7 / tyrosinase cells. Expression of transduced tyrosinase was evaluated by flow cytometry after staining with an anti-tyrosinase monoclonal antibody (mAb) (clone ERP10141; Abeam). HLA-C*05:01 -Malme-3M and Me275 cells to generate Malme-3M / C*05:01 and Me275 / C*05:01 cells. The HLA-C*05:01 gene was tagged with the ΔNGFR gene as described above and ΔNGFR + Cells were purified (>95% purity) and used for subsequent experiments. The ΔNGFR gene alone was retrovirally transduced as a control.

[0373] MAGE-A1 cells were retrovirally transduced with the full-length MAGE-A1 gene - SK-MEL-21 cells to generate SK-MEK-21 / MAGE-A1 cells. Expression of transduced MAGE-A1 was evaluated by flow cytometry after staining with anti-MAGE-A1 mAb (clone MA454; LifeSpan Biosciences). HLA-B*07:02 - Me275 and SK-MEL-37 cells to generate Me275 / B*07:02 and SK-MEL-37 / B*07:02 cells. The HLA-B*07:02 gene was tagged with the ΔNGFR gene as described above and ΔNGFR + Cells were purified (>95% purity) and used for subsequent experiments. The ΔNGFR gene alone was retrovirally transduced as a control.

[0374] MART1 cells were retrovirally transduced with the full-length MART1 gene - A375 cells to generate A375 / MART1 cells. Expression of transduced MART1 was evaluated by flow cytometry after staining with anti-MART1 mAb (clone A103; Santa Cruz Biotechnology). HLA-B*18:01 - Malme-3M, SK-MEL-28 and A375 cells to generate Malme-3M / B*18:01, SK-MEL-28 / B*18:01 and A375 / B*18:01 cells. The HLA-B*18:01 gene was tagged with the ΔNGFR gene as described above and ΔNGFR + Cells were purified (>95% purity) and used for subsequent experiments. The ΔNGFR gene alone was retrovirally transduced as a control.

[0375] MAGE-A3 cells were retrovirally transduced with the full-length MAGE-A3 gene -HEK293T cells were used to generate HEK293T / MAGE-A3 cells. MAGE-A3 expression in transduced cells was evaluated by Western blotting using an anti-MAGE-A3 polyclonal antibody (pAb) (LifeSpan Biosciences). HLA-B*18:01 was transduced retrovirally using HLA-B*18:01. - SK-MEL-28 and HEK293T cells were used to generate SK-MEL-28 / B*18:01 and HEK293T / B*18:01 cells. The HLA-B*18:01 gene was tagged with the ΔNGFR gene as described above, and the ΔNGFR... + Cells were purified (>95% purity) and used in subsequent experiments. The ΔNGFR gene alone was transduced retrovirally as a control.

[0376] SSX2 was transduced using the full-length SSX2 gene via retrovirus. - SK-MEL-21 and SK-MEL-28 cells were used to generate SK-MEK-21 / SSX2 and SK-MEL-28 / SSX2 cells. SSX2 expression in transduced cells was evaluated by Western blotting using anti-SSX2 pAb (Thermo Fisher Scientific). HLA-A*02:01 was transduced retrovirally using HLA-A*02:01. - SK-MEL-28 cells were used to generate SK-MEL-28 / A*02:01 cells.

[0377] Flow cytometry and cell sorting

[0378] Cell surface molecules were stained using PC5-conjugated anti-CD8 mAb (clone B9.11; Beckman Coulter), FITC-conjugated anti-NGFR (clone ME20.4; Biolegend), and APC / Cy7-conjugated anti-CD3 (clone UCHT1; Biolegend). Dead cells were identified using the LIVE / DEAD Fixable Aqua Cell Staining Kit (Life Technologies). For intracellular staining, cells were fixed and permeabilized using the Cytofix / Cytoperm Kit (BD Biosciences). Stained cells were analyzed by flow cytometry (BD Biosciences), and data analysis was performed using FlowJo (Tree Star). Cell sorting was performed using FACS Aria II (BD Biosciences).

[0379] Cytokine ELISPOT Analysis

[0380] IFN-γ ELISPOT assay was performed as previously described (see, e.g., Kagoya et al., Nat. Commun. 9:1915 (2018); Anczurowski et al., Sci. Rep. 8:4804 (2018); and Yamashita et al., Nat Commun. 8: 15244 (2017)). PVDF plates (Millipore, Bedford, MA) were coated with a capture mAb (1-D1K; MABTECH, Mariemont, OH) and T cells were incubated with 2 x 105 4 target cells per well in the presence or absence of peptide at 37°C for 20-24 hours. Plates were then washed and incubated with a biotin-conjugated detection mAb (7-B6-1; MABTECH). HRP-conjugated SA (Jackson ImmunoResearch) was then added and IFN-γ spots were developed. The reaction was stopped by a thorough rinse with cold tap water. ELISPOT plates were scanned and counted using an ImmunoSpot plate reader and ImmunoSpot version 5.0 software (Cellular Technology Limited, Shaker Heights, OH).

[0381] CD8 + Expansion of TIL in an HLA-restricted peptide-specific manner

[0382] As applicable, e.g., for MAGE-A3 TCR, CD8 + T cells were purified by negative magnetic selection using CD8 + TIL. B*18:01-artificial APCs were pulsed with 10 pg / mL of the class I restricted peptide of interest for 6 hours. Artificial APCs were then irradiated at 200 Gy, washed, and added to TIL at an effector to target (E:T) ratio of 20:1. From the next day, 10 IU / ml IL-2 (Novartis), 10 ng / ml IL-15 (Peprotech), and 30 ng / ml IL-21 (Peprotech) were added to the cultures every three days.

[0383] Expansion of primary CD8 + T cells transduced with cloned TCR

[0384] CD3 +T cells. Purified T cells were stimulated with 200 Gy irradiated artificial APCs / mOKT3 at an E:T ratio of 20:1. From the next day, activated T cells were transduced with cloned TCR genes in retrovirus via centrifugation at 1,000 g for 1 hour at 32°C for 3 consecutive days. The next day, 100 IU / ml IL-2 and 10 ng / ml IL-15 were added to the TCR transduced T cells. Medium was replenished every 2-3 days.

[0385] Generation of pHLA multimers based on mammalian cells

[0386] Engineered HLA class I genes with affinity matured HLA class I alpha 2 domains and replacement of HLA class I alpha 3 domains with mouse K b The position 115 of the genetically derived alpha 3 domain carries a Glu (E) residue instead of a Gin (Q) residue. Soluble HLA class I Q115E -K b genes were generated by fusing the extracellular domain of affinity matured HLA class I genes with a Gly-Ser (GS) flexible linker followed by a 6x His tag. HEK293T cells were transduced individually with various soluble HLA class I Q115E -K b genes together with the beta 2m gene using a retroviral system based on 293GPG cells. Stable HEK293T cells ectopically expressing soluble affinity matured class I Q115E -K b were grown until confluency and then the medium was exchanged. Forty-eight hours later, the conditioned medium was harvested and immediately used or frozen until use. Supernatants produced by HEK293T transfectants containing soluble HLA class I Q115E -K b were incubated with 100-1000 pg / ml of the class I restricted peptide of interest at 37°C overnight to achieve in vitro peptide exchange. Peptide loaded soluble monomeric class I Q115E -K b was dimerized using an anti-His mAb (clone AD1.1.10; Abeam) conjugated to a fluorescent dye such as phycoerythrin (PE) at a 2:1 molar ratio for 2 hours at room temperature or overnight at 4°C. The concentration of functional soluble HLA class I Q115E -K b molecules was measured by specific ELISA using an anti-pan class I mAb (clone W6 / 32, in-house) and an anti-His tag biotinylated mAb (clone AD1.1.10, R&D systems) as capture and detection Ab, respectively.

[0387] pHLA multimer staining

[0388] T cells (1 x 105) were incubated for 30 minutes at 37°C in the presence of 50 nM dasatinib (LC laboratories). Cells were then washed and incubated with 5-10 pg / ml of multimer for 30 minutes at room temperature and 15 minutes at 4°C with R-phycoerythrin-conjugated AffiniPure Fab fragment goat anti-mouse IgGl (Jackson ImmunoResearch Laboratories) added. Next, cells were washed three times and co-stained with anti-CD8 mAb for 15 minutes at 4°C. Finally, LIVE / DEAD fixable dead cell stain kit was used to discriminate dead cells. 5

[0389] Statistical analysis

[0390] Statistical analysis was performed using GraphPad Prism 5.0e. Welch's t-test (two-sided) analysis was performed to determine if two groups were significantly different for a given variable. P values < 0.05 were considered significant.

[0391] Example 2 - Tyrosinase-specific TCR

[0392] Tumor infiltrating lymphocytes (TILs) were isolated from a metastatic melanoma patient and then polyclonally expanded in vitro and examined for tyrosinase antigen specificity to the HLA-C*05:01 allele. A combination of structure-based analysis using peptide / HLA (pHLA) multimers and functional analysis was used to measure antigen-specific T cell responses. pHLA multimers with tyrosinase 460-468 peptides stained T cells (Figure 1). TILs showed positivity to C*05:01 / tyrosinase 460-468 multimers. According to ELISPOT analysis, multimer-positive T cells secreted detectable IFN-γ Figure 2 ) in an HLA-restricted peptide-specific manner.

[0393] Multimer-positive anti-tumor T cells were collected and their TCR genes were molecularly cloned (Figure 3). Antigen specificity and functional reactivity of cloned TCRs were verified by multimer staining and ELISPOT assays of TCR-reconstituted T cells. When reconstituted on primary T cells, C*05:01 / tyrosinase 460-468 TCR-transduced T cells successfully stained with cognate multimers (Figure 4) and strongly reacted 460-468 with tyrosinase Figure 5 ​). Importantly, these cells were able to recognize C*05:01 -matched and unpeptide pulsed tumor cells that naturally express the tyrosinase gene. While the Malme-3M and Me275 melanoma cell lines are negative for C*05:01, they endogenously express the tyrosinase gene. When C*05:01 molecules were ectopically expressed, C*05:01 / tyrosinase 460-468 TCR-transduced T cells successfully recognized two melanoma cell lines. In addition, MCF7 breast cancer cells, which lack endogenous expression of tyrosinase, became C*05:01 / tyrosinase 460-468 TCR-transduced T cells were reactive (Figures 6-8). These results clearly demonstrate that C*05:01 / tyrosinase 460-468 TCR-transduced T cells were of sufficient avidity to recognize tumor cells and C*05:01 / tyrosinase 460-468 TCRs were tumor reactive.

[0394] Example 3 - MAGE-A1 -specific TCRs

[0395] TILs were isolated from a metastatic melanoma patient and then polyclonally expanded in vitro and examined for MAGE-A1 antigen specificity to the HLA-B*07:02 allele. Using a pHLA multimer with MAGE-A1 289-297 peptide (Figure 9). The TILs showed positive staining for the B*07:02 / MAGE-A1 289-297 multimer. According to ELISPOT analysis, multimer-positive T cells secreted detectable IFN-γ Figure 10 ) in an HLA-restricted peptide-specific manner.

[0396] Multimer-positive antitumor T cells were collected and their TCR genes were molecularly cloned (Figure 11). The antigen specificity and functional reactivity of the cloned TCRs were verified by multimer staining and ELISPOT assays of TCR-reconstituted T cells. When reconstituted on primary T cells, the B*07:02 / MAGE-A1 289-297 TCR-transduced T cells were successfully stained with the cognate multimer (Figure 12) and strongly reacted with MAGE-A1 289-297 peptide presented by surface B*07:02 molecules Figure 13). Importantly, these cells were able to recognize B*07:02 matched and non-peptide pulsed tumor cells that naturally express the MAGE-A1 gene. While both Me275 and SK-MEL-37 melanoma cell lines are negative for B*07:02, they endogenously express the MAGE-A1 gene. When the B*07:02 molecule is ectopically expressed, the B*07:02 / MAGE-A1 289-297 TCR transduced T cells successfully recognized both melanoma cell lines. In addition, SK-MEL-21 melanoma cells, which lack endogenous expression of MAGE-A1, became recognized by B*07:02 / MAGE-A1 289-297 TCR transduced T cells were reactive (Figures 14-16). These results clearly demonstrate that B*07:02 / MAGE-A1 289-297 TCR transduced T cells were of sufficient avidity to recognize tumor cells and the cloned B*07:02 / MAGE-A1 289-297 TCRs were tumor reactive.

[0397] The use of newly cloned tumor reactive B*07:02 restricted MAGE-A1 TCR genes can expand the applicability of anti-MAGE-A1 TCR gene therapy.

[0398] Example 4 - MART1 specific TCRs

[0399] TILs were isolated from a metastatic melanoma patient and then polyclonally expanded in vitro and examined for their MART1 antigen specificity for the HLA-B*18:01 allele. Since the generation of pHLA multimers requires the use of peptides with known accurate sequences, the use of a pHLA multimer based strategy for high throughput screening of novel epitope peptides is neither simple nor practical. In addition to structure based analysis using pHLA multimers, functional analysis can also be applied to determine the antigen specificity of T cells. Functional assays were performed using artificial antigen presenting cells (APCs) as stimulator cells that can take up and process longer peptides and present the epitope peptides via class I molecules. HLA-B*18:01 - artificial APCs were pulsed with overlapping peptides covering the entire protein of MART1 (Table 5) and used as stimulators in a cytokine ELISPOT assay. In the IFN-γ ELISPOT analysis, B*18:01 + Melanoma TILs showed positive reactivity for two adjacent peptides with the consensus sequence 21 YTTAEEAAGIGILTV 35 Figure 17 ​). Using a series of mutant deletion peptides, the minimal required peptide epitope presented by the B*18:01 molecule was determined 25 EEAAGIGIL 33 Importantly, B*18:01 / MART1 25-33 Multimers successfully stained up to 9.2% of polyclonally expanded TILs, indicating that B*18:01 / MART1 25-33 T cells were the dominant population of TILs (Figure 18). According to ELISPOT analysis, multimer-positive T cells secreted detectable IFN-γ Figure 19 ) in an HLA-restricted peptide-specific manner.

[0400] Table 5. MART1 -derived overlapping peptides.

[0401] Position Peptide sequence SEQ ID NO 1 MPREDAHFIYGYPKKGHGHS 61 6 AHFIYGYPKKGHGHSYTTAE 62 11 GYPKKGHGHSYTTAEEAAGI 63 16 GHGHSYTTAEEAAGIGILTV 64 21 YTTAEEAAGIGILTVILGVL 65 26 EAAGIGILTVILGVLLLIGC 66 31 GILTVILGVLLLIGCWYCRR 67 36 ILGVLLLIGCWYCRRRNGYR 68 41 LLIGCWYCRRRNGYRALMDK 69 46 WYCRRRNGYRALMDKSLHVG 70 51 RNGYRALMDKSLHVGTQCAL 71 56 ALMDKSLHVGTQCALTRRCP 72 61 SLHVGTQCALTRRCPQEGFD 73 66 TQCALTRRCPQEGFDHRDSK 74 71 TRRCPQEGFDHRDSKVSLQE 75 76 QEGFDHRDSKVSLQEKNCEP 76 81 HRDSKVSLQEKNCEPVVPNA 77 86 VSLQEKNCEPVVPNAPPAYE 78 91 KNCEPVVPNAPPAYEKLSAE 79 96 VVPNAPPAYEKLSAEQSPPP 80 99 NAPPAYEKLSAEQSPPPYSP 81

[0402] Multimer-positive anti-tumor T cells were collected and the TCR genes were molecularly cloned (Figure 20). The antigen specificity and functional reactivity of the cloned TCRs were verified by multimer staining and ELISPOT assays of TCR- reconstituted T cells. When reconstituted on primary T cells, B*18:01 / MART1 25-33 TCR-transduced T cells successfully stained with the cognate multimer (Figure 21) and reacted strongly with MART1 25-33 peptides presented by surface B*18:01 molecules. Figure 22 Importantly, these cells were able to recognize B*18:01 -matched tumor cells that naturally express the MART1 gene and were not pulsed with peptides. While both Malme-3M and SK-MEL-28 melanoma cell lines are negative for B*18:01, they endogenously express the MART1 gene. When B*18:01 molecules are ectopically expressed, B*18:01 / MART1 25-33 TCR-transduced T cells successfully recognized both melanoma cell lines. In addition, A375 melanoma cells, which lack endogenous expression of both B*18:01 and MART1, became susceptible to B*18:01 / MART1 25- 33 TCR-transduced T cells were reactive (Figures 23-25). These results clearly demonstrate that B*18:01 / MART1 25-33 TCR-transduced T cells were of sufficient avidity to recognize tumor cells and the cloned B*18:01 / MART1 25-33 TCR was tumor-reactive.

[0403] The use of newly cloned tumor-reactive B*18:01 -restricted MART1 TCR genes can expand the applicability of anti-MART1 TCR gene therapy beyond HLA-A*02:01 -positive cancer patients.

[0404] Example 5 - MAGE-A3-specific TCR

[0405] TILs were isolated from a metastatic melanoma patient, then polyclonally expanded in vitro and examined for MAGE-A3 antigen specificity to the HLA-B*18:01 allele. Using a pHLA multimer with the MAGE-A3 167-176 peptide, T cells were stained. Prior to peptide-specific stimulation, B*18:01 / MAGE-A3 167-176 multimer positivity was only 0.04%. However, when the TILs were weakly stimulated by B*18:01 - artificial APCs pulsed with MAGE-A3 167-176 peptide, 5.5% of the TILs stained with the cognate multimer and secreted IFN-γ in a B*18:01 / MAGE-A3 167-176 specific manner (Figures 26 to Figure 27 ).

[0406] Multimer-positive anti-tumor T cells were collected and their TCR genes were molecularly cloned (Figure 28). The antigen specificity and functional reactivity of the cloned TCRs were verified by multimer staining and ELISPOT assays of TCR-reconstituted T cells. When reconstituted on primary T cells, B*18:01 / MAGE-A3 167-176 TCR-transduced T cells successfully stained with the cognate multimer (Figure 29) and strongly reacted with MAGE-A3 167-176 peptide presented by surface B*18:01 molecules ( Figure 30 ). Importantly, these cells were able to recognize B*18:01 -matched tumor cells that naturally express the MAGE-A3 gene without peptide pulsing. While SK-MEL-28 melanoma cells are negative for B*18:01, they endogenously express the MAGE-A3 gene. When B*18:01 molecules are ectopically expressed, HEK293T melanoma cells that lack endogenous expression of both B*18:01 and MAGE-A3 become recognized by B*18:01 / MAGE-A3 167-176 TCR-transduced T cells successfully recognized melanoma cells. In addition, HEK293T melanoma cells that lack endogenous expression of both B*18:01 and MAGE-A3 became recognized by B*18:01 / MAGE-A3 167-176TCR-transduced T cells were reactive (Figures 31-33). These results clearly demonstrate that B*18:01 / MAGE-A3 167-176 TCR-transduced T cells were of sufficient avidity to recognize tumor cells and were clonally expanded B*18:01 / MAGE-A3 167-176 TCRs were tumor-reactive.

[0407] The use of newly cloned tumor-reactive B*18:01 / MAGE-A3 TCR genes can expand the applicability of anti-MAGE-A3 TCR gene therapy beyond HLA-A*02:01 positive cancer patients.

[0408] Example 6 - SSX2-specific TCR

[0409] TILs were isolated from a metastatic melanoma patient, then polyclonally expanded in vitro, and examined for SSX2 antigen specificity to the HLA-A*02:01 allele. Using pHLA multimers with SSX2 41-49 peptides, T cells were stained (Figure 34). TILs showed positive to A*02:01 / SSX2 41-49 multimers. According to ELISPOT analysis, multimer-positive T cells secreted detectable IFN-γ Figure 35 ) in an HLA-restricted peptide-specific manner.

[0410] Multimer-positive anti-tumor T cells were collected and their TCR genes were molecularly cloned (Figure 36). Antigen specificity and functional reactivity of the cloned TCRs were verified by multimer staining and ELISPOT assays of TCR-reconstituted T cells. When reconstituted on primary T cells, A*02:01 / SSX2 41-49 TCR-transduced T cells successfully stained with cognate multimers (Figure 37) and strongly reacted with SSX2 41-49 peptides presented by surface A*02:01 molecules Figure 38 . Importantly, these cells were able to recognize A*02:01 -matched and unpeptide-pulsed tumor cells that naturally express the SSX2 gene. While SK-MEL-21 melanoma cells are positive for A*02:01, they do not endogenously express the SSX2 gene. When the SSX2 molecule is ectopically expressed, SK-MEL-21 cells become positive for A*02:01 / SSX2 41-49 TCR-transduced T cells successfully recognized melanoma cells. In addition, SK-MEL-28 melanoma cells, which lack endogenous expression of both A*02:01 and SSX2, became positive for A*02:01 / SSX2 41-49TCR-transduced T cells were reactive (Figures 39-41). These results clearly demonstrate that A*02:01 / SSX2 41-49 TCR-transduced T cells were of sufficient avidity to recognize tumor cells and clonal A*02:01 / SSX2 41-49 TCRs were tumor-reactive.

[0411] Use of newly cloned tumor-reactive A*02:01 restricted SSX2 TCR genes can expand the applicability of anti-SSX2 TCR gene therapy. SEQUENCE LISTING <110> University Health Network <120> T cell receptors and methods of use thereof <130> 4285.008PC01 / C-K / BMD <150> US 62 / 823,487 <151> 2019-03-25 <160> 93 <170> PatentIn version 3.5 <210> 1 <211> 271 <212> PRT <213> Artificial Sequence <220> <223> Tyr-alpha chain amino acid sequence <400> 1 Met Arg Gin Val Ala Arg Val lie Val Phe Leu Thr Leu Ser Thr Leu 1 5 10 15 Ser Leu Ala Lys Thr Thr Gin Pro lie Ser Met Asp Ser Tyr Glu Gly 20 25 30 Gln Glu Val Asn lie Thr Cys Ser His Asn Asn lie Ala Thr Asn Asp 35 40 45 Tyr lie Thr Trp Tyr Gin Gin Phe Pro Ser Gin Gly Pro Arg Phe lie 50 55 60 Ile Gin Gly Tyr Lys Thr Lys Val Thr Asn Glu Val Ala Ser Leu Phe 65 70 75 80 Ile Pro Ala Asp Arg Lys Ser Ser Thr Leu Ser Leu Pro Arg Val Ser 85 90 95 Leu Ser Asp Thr Ala Val Tyr Tyr Cys Leu Val Gly Asp Val Glu Gly 100 105 110 Ser Gin Gly Asn Leu Ile Phe Gly Lys Gly Thr Lys Leu Ser Val Lys 115 120 125 Pro Asn Ile Gin Asn Pro Asp Pro Ala Val Tyr Gin Leu Arg Asp Ser 130 135 140 Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gin 145 150 155 160 Thr Asn Val Ser Gin Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys 165 170 175 Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val 180 185 190 Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn 195 200 205 Ser Ile Ile Pro Gin Asp Thr Phe Phe Pro Ser Pro Gin Ser Ser Cys 210 215 220 Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn 225 230 235 240 Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val 245 250 255 Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser Glx 260 265 270 <210> 2 <211> 314 <212> PRT <213> Artificial Sequence <220> <223> Tyr-beta chain amino acid sequence <400> 2 Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser His His Ser Gly Gly Ile Tyr Asn Glu Gln Phe Phe Gly Pro Gly 115 120 125 Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu 130 135 140 Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr 180 185 190 Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr 195 200 205 Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro 210 215 220 Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn 225 230 235 240 Asp Glu Trp Thr Gin Asp Arg Ala Lys Pro Val Thr Gin He Val Ser 245 250 255 Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr 260 265 270 Gln Gin Gly Val Leu Ser Ala Thr He Leu Tyr Glu He Leu Leu Gly 275 280 285 Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala 290 295 300 Met Val Lys Arg Lys Asp Ser Arg Gly Glx 305 310 <210> 3 <211> 813 <212> DNA <213> Artificial Sequence <220> <223> Tyr-alpha chain nucleotide sequence <400> 3 atgaggcaag tggcgagagt gatcgtgttc ctgaccctga gtactttgag ccttgctaag 60 accacccagc ccatctccat ggactcatat gaaggacaag aagtgaacat aacctgtagc 120 cacaacaaca ttgctacaaa tgattatatc acgtggtacc aacagtttcc cagccaagga 180 ccacgattta ttattcaagg atacaagaca aaagttacaa acgaagtggc ctccctgttt 240 atccctgccg acagaaagtc cagcactctg agcctgcccc gggtttccct gagcgacact 300 gctgtgtact actgcctcgt gggtgacgta gaaggaagcc aaggaaatct catctttgga 360 aaaggcacta aactctctgt taaaccaaat atccagaacc ctgaccctgc cgtgtaccag 420 ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 480 acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 540 atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 600 gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 660 gaaagttcct gtgatgtcaa gctggtcgag aaaagctttg aaacagatac gaacctaaac 720 tttcaaaacc tgtcagtgat tgggttccga atcctcctcc tgaaagtggc cgggtttaat 780 ctgctcatga cgctgcggct gtggtccagc tga 813 <210> 4 <211> 942 <212> DNA <213> Artificial Sequence <220> <223> Tyr-beta Chain Nucleotide Sequence <400> 4 atgagcatcg gcctcctgtg ctgtgcagcc ttgtctctcc tgtgggcagg tccagtgaat 60 gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 120 cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 180 gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 240 aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 300 gctccctccc agacatctgt gtacttctgt gccagcagtc accattcggg gggatctac 360 aatgagcagt tcttcgggcc agggacacgg ctcaccgtgc tagaggacct gaaaaacgtg 420 ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480 gccacactgg tatgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 540 gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600 cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660 tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720 gacgagtgga cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg 780 ggtagagcag actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc 840 atcctctatg agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc 900 gtgctgatgg ccatggtcaa gagaaaggat tccagaggct ag 942 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Tyr-alpha CDR1 <400> 5 Asn lie Ala Thr Asn Asp Tyr 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Tyr-alpha CDR2 <400> 6 Gly Tyr Lys Thr Lys 1 5 <210> 7 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Tyr-alpha CDR3 <400> 7 Cys Leu Val Gly Asp Val Glu Gly Ser Gin Gly Asn Leu lie Phe 1 5 10 15 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Tyr-beta CDR1 <400> 8 Met Asn His Glu Tyr 1 5 <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Tyr-beta CDR2 <400> 9 Ser Val Gly Ala Gly Ile 1 5 <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Tyr-beta CDR3 <400> 10 Cys Ala Ser Ser His His Ser Gly Gly Ile Tyr Asn Glu Gin Phe Phe 1 5 10 15 <210> 11 <211> 280 <212> PRT <213> Artificial Sequence <220> <223> MAGE-A1-alpha chain amino acid sequence <400> 11 Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val 1 5 10 15 Val Ser Ser Met Ala Gin Lys Val Thr Gin Ala Gin Thr Glu He Ser 20 25 30 Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg 35 40 45 Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gin Pro Pro Ser Gly Glu 50 55 60 Leu Val Phe Leu He Arg Arg Asn Ser Phe Asp Glu Gin Asn Glu He 65 70 75 80 Ser Gly Arg Tyr Ser Trp Asn Phe Gin Lys Ser Thr Ser Ser Phe Asn 85 90 95 Phe Thr He Thr Ala Ser Gin Val Val Asp Ser Ala Val Tyr Phe Cys 100 105 110 Ala Leu Ser Glu Ser Tyr Ser Gly Ala Gly Ser Tyr Gin Leu Thr Phe 115 120 125 Gly Lys Gly Thr Lys Leu Ser Val He Pro Asn He Gin Asn Pro Asp 130 135 140 Pro Ala Val Tyr Gin Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val 145 150 155 160 Cys Leu Phe Thr Asp Phe Asp Ser Gin Thr Asn Val Ser Gin Ser Lys 165 170 175 Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser 180 185 190 Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp 195 200 205 Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr 210 215 220 Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys 225 230 235 240 Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile 245 250 255 Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met 260 265 270 Thr Leu Arg Leu Trp Ser Ser Glx 275 280 <210> 12 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> MAGE-A1 -beta chain amino acid sequence <400> 12 Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala 1 5 10 15 Asp His Ala Asp Thr Gly Val Ser Gin Asn Pro Arg His Lys He Thr 20 25 30 Lys Arg Gly Gin Asn Val Thr Phe Arg Cys Asp Pro He Ser Glu His 35 40 45 Asn Arg Leu Tyr Trp Tyr Arg Gin Thr Leu Gly Gin Gly Pro Glu Phe 50 55 60 Leu Thr Tyr Phe Gin Asn Glu Ala Gin Leu Glu Lys Ser Arg Leu Leu 65 70 75 80 Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu 85 90 95 Glu He Gin Arg Thr Glu Gin Gly Asp Ser Ala Met Tyr Leu Cys Ala 100 105 110 Ser Ser Leu Ala Ser Gly Ser Asn Gin Pro Gin His Phe Gly Asp Gly 115 120 125 Thr Arg Leu Ser He Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu 130 135 140 Val Ala Val Phe Glu Pro Ser Glu Ala Glu He Ser His Thr Gin Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr 180 185 190 Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr 195 200 205 Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro 210 215 220 Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn 225 230 235 240 Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser 245 250 255 Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr 260 265 270 Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly 275 280 285 Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala 290 295 300 Met Val Lys Arg Lys Asp Phe Glx 305 310 <210> 13 <211> 840 <212> DNA <213> Artificial Sequence <220> <223> MAGE-A1-alpha chain nucleotide sequence <400> 13 atgctgactg ccagcctgtt gagggcagtc atagcctcca tctgtgttgt atccagcatg 60 gctcagaagg taactcaagc gcagactgaa atttctgtgg tggagaagga ggatgtgacc 120 ttggactgtg tgtatgaaac ccgtgatact acttattact tattctggta caagcaacca 180 ccaagtggag aattggtttt ccttattcgt cggaactctt ttgatgagca aaatgaaata 240 agtggtcggt attcttggaa cttccagaaa tccaccagtt ccttcaactt caccatcaca 300 gcctcacaag tcgtggactc agcagtatac ttctgtgctc tgagtgagtc atactctggg 360 gctgggagtt accaactcac tttcgggaag gggaccaaac tctcggtcat accaaatatc 420 cagaaccctg accctgccgt gtaccagctg agagactcta aatccagtga caagtctgtc 480 tgcctattca ccgattttga ttctcaaaca aatgtgtcac aaagtaagga ttctgatgtg 540 tatatcacag acaaaactgt gctagacatg aggtctatgg acttcaagag caacagtgct 600 gtggcctgga gcaacaaatc tgactttgca tgtgcaaacg ccttcaacaa cagcattatt 660 ccagaagaca ccttcttccc cagcccagaa agttcctgtg atgtcaagct ggtcgagaaa 720 agctttgaaa cagatacgaa cctaaacttt caaaacctgt cagtgattgg gttccgaatc 780 ctcctcctga aagtggccgg gtttaatctg ctcatgacgc tgcggctgtg gtccagctga 840 <210> 14 <211> 936 <212> DNA <213> Artificial Sequence <220> <223> MAGE-A1 -beta chain nucleotide sequence <400> 14 atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60 actggagtct cccagaaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120 aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180 ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240 agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300 acagagcagg gggactcggc catgtatctc tgtgccagca gcttagcttc gggcagcaat 360 cagccccagc attttggtga tgggactcga ctctccatcc tagaggacct gaacaaggtg 420 ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480 gccacactgg tgtgcctggc cacaggcttc ttccccgacc acgtggagct gagctggtgg 540 gtgaatggga aggaggtgca cagtggggtc agcacggacc cgcagcccct caaggagcag 600 cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660 tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720 gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 780 ggtagagcag actgtggctt tacctcggtg tcctaccagc aaggggtcct gtctgccacc 840 atcctctatg agatcctgct agggaaggcc accctgtatg ctgtgctggt cagcgccctt 900 gtgttgatgg ccatggtcaa gagaaaggat ttctga 936 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MAGE-A1-alpha CDR1 <400> 15 Thr Arg Asp Thr Thr Tyr Tyr Leu 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MAGE-A1-alpha CDR1 <400> 16 Arg Asn Ser Phe Asp Glu Gin Asn 1 5 <210> 17 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> MAGE-A1-alpha CDR2 <400> 17 Cys Ala Leu Ser Glu Ser Tyr Ser Gly Ala Gly Ser Tyr Gin Leu Thr 1 5 10 15 Phe <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> MAGE-A1-beta CDR1 <400> 18 Ser Glu His Asn Arg 1 5 <210> 19 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> MAGE-A1-beta CDR2 <400> 19 Phe Gin Asn Glu Ala Gin 1 5 <210> 20 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> MAGE-A1 -beta CDR3 <400> 20 Cys Ala Ser Ser Leu Ala Ser Gly Ser Asn Gln Pro Gln His Phe 1 5 10 15 <210> 21 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> MART1 -alpha chain amino acid sequence <400> 21 Met Trp Gly Val Phe Leu Leu Tyr Val Ser Met Lys Met Gly Gly Thr 1 5 10 15 Thr Gly Gln Asn Ile Asp Gln Pro Thr Glu Met Thr Ala Thr Glu Gly 20 25 30 Ala Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Asn Gly 35 40 45 Leu Phe Trp Tyr Gln Gln His Ala Gly Glu Ala Pro Thr Phe Leu Ser 50 55 60 Tyr Asn Val Leu Asp Gly Leu Glu Glu Lys Gly Arg Phe Ser Ser Phe 65 70 75 80 Leu Ser Arg Ser Lys Gly Tyr Ser Tyr Leu Leu Leu Lys Glu Leu Gln 85 90 95 Met Lys Asp Ser Ala Ser Tyr Leu Cys Ala Val Tyr Gly Gly Ala Thr 100 105 110 Asn Lys Leu Ile Phe Gly Thr Gly Thr Leu Leu Ala Val Gln Pro Asn 115 120 125 Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser 130 135 140 Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn 145 150 155 160 Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val 165 170 175 Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp 180 185 190 Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile 195 200 205 Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val 210 215 220 Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln 225 230 235 240 Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly 245 250 255 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser Glx 260 265 <210> 22 <211> 313 <212> PRT <213> Artificial Sequence <220> <223> MART1 -beta chain amino acid sequence <400> 22 Met Asp Thr Arg Val Leu Cys Cys Ala Val Ile Cys Leu Leu Gly Ala 1 5 10 15 Gly Leu Ser Asn Ala Gly Val Met Gln Asn Pro Arg His Leu Val Arg 20 25 30 Arg Arg Gly Gln Glu Ala Arg Leu Arg Cys Ser Pro Met Lys Gly His 35 40 45 Ser His Val Tyr Trp Tyr Arg Gln Leu Pro Glu Glu Gly Leu Lys Phe 50 55 60 Met Val Tyr Leu Gln Lys Glu Asn Ile Ile Asp Glu Ser Gly Met Pro 65 70 75 80 Lys Glu Arg Phe Ser Ala Glu Phe Pro Lys Glu Gly Pro Ser Ile Leu 85 90 95 Arg Ile Gln Gln Val Val Arg Gly Asp Ser Ala Ala Tyr Phe Cys Ala 100 105 110 Ser Ser Pro His Ala Gly Gly Val Asp Glu Lys Leu Phe Phe Gly Ser 115 120 125 Gly Thr Gln Leu Ser Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro 130 135 140 Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln 145 150 155 160 Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val 165 170 175 Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 180 185 190 Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg 195 200 205 Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 210 215 220 Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu 225 230 235 240 Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val 245 250 255 Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser 260 265 270 Tyr Gin Gin Gly Val Leu Ser Ala Thr He Leu Tyr Glu He Leu Leu 275 280 285 Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met 290 295 300 Ala Met Val Lys Arg Lys Asp Phe Glx 305 310 <210> 23 <211> 807 <212> DNA <213> Artificial Sequence <220> <223> MART1-alpha chain nucleotide sequence <400> 23 atgtggggag ttttccttct ttatgtttcc atgaagatgg gaggcactac aggacaaaac 60 attgaccagc ccactgagat gacagctacg gaaggtgcca ttgtccagat caactgcacg 120 taccagacat ctgggttcaa cgggctgttc tggtaccagc aacatgctgg cgaagcaccc 180 acatttctgt cttacaatgt tctggatggt ttggaggaga aaggtcgttt ttcttcattc 240 cttagtcggt ctaaagggta cagttacctc cttttgaagg agctccagat gaaagactct 300 gcctcttacc tctgtgctgt gtatggtggt gctacaaaca agctcatctt tggaactggc 360 Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Metactctgcttg ctgtccagcc aaatatccag aaccctgacc ctgccgtgta ccagctgaga 420 gactctaaat ccagtgacaa gtctgtctgc ctattcaccg attttgattc tcaaacaaat 480 gtgtcacaaa gtaaggattc tgatgtgtat atcacagaca aaactgtgct agacatgagg 540 tctatggact tcaagagcaa cagtgctgtg gcctggagca acaaatctga ctttgcatgt 600 gcaaacgcct tcaacaacag cattattcca gaagacacct tcttccccag cccagaaagt 660 tcctgtgatg tcaagctggt cgagaaaagc tttgaaacag atacgaacct aaactttcaa 720 aacctgtcag tgattgggtt ccgaatcctc ctcctgaaag tggccgggtt taatctgctc 780 atgacgctgc ggctgtggtc cagctga 807 <210> 24 <211> 939 <212> DNA <213> Artificial Sequence <220> <223> MART1 -beta chain nucleotide sequence <400> 24 atggacacca gagtactctg ctgtgcggtc atctgtcttc tgggggcagg tctctcaaat 60 gccggcgtca tgcagaaccc aagacacctg gtcaggagga ggggacagga ggcaagactg 120 agatgcagcc caatgaaagg acacagtcat gtttactggt atcggcagct cccagaggaa 180 ggtctgaaat tcatggttta tctccagaaa gaaaatatca tagatgagtc aggaatgcca 240 aaggaacgat tttctgctga atttcccaaa gagggcccca gcatcctgag gatccagcag 300 gtagtgcgag gagattcggc agcttatttc tgtgccagct caccacacgc ggggggagtt 360 gatgaaaaac tgttttttgg cagtggaacc cagctctctg tcttggagga cctgaacaag 420 gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag cagagatctc ccacacccaa 480 aaggccacac tggtgtgcct ggccacaggc ttcttccctg accacgtgga gctgagctgg 540 tgggtgaatg ggaaggaggt gcacagtggg gtcagcacgg acccgcagcc cctcaaggag 600 cagcccgccc tcaatgactc cagatactgc ctgagcagcc gcctgagggt ctcggccacc 660 ttctggcaga acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 720 aatgacgagt ggacccagga tagggccaaa cccgtcaccc agatcgtcag cgccgaggcc 780 tggggtagag cagactgtgg ctttacctcg gtgtcctacc agcaaggggt cctgtctgcc 840 accatcctct atgagatcct gctagggaag gccaccctgt atgctgtgct ggtcagcgcc 900 cttgtgttga tggccatggt caagagaaag gatttctga 939 <210> 25 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> MART1-alpha CDR1 <400> 25 Thr Ser Gly Phe Asn Gly 1 5 <210> 26 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> MART1-alpha CDR2 <400> 26 Asn Val Leu Asp Gly Leu 1 5 <210> 27 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> MART1-alpha CDR3 <400> 27 Cys Ala Val Tyr Gly Gly Ala Thr Asn Lys Leu Ile Phe 1 5 10 <210> 28 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> MART1-beta CDR1 <400> 28 Lys Gly His Ser His 1 5 <210> 29 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> MART1-beta CDR2 <400> 29 Leu Gln Lys Glu Asn Ile 1 5 <210> 30 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> MART1-beta CDR3 <400> 30 Cys Ala Ser Ser Pro His Ala Gly Gly Val Asp Glu Lys Leu Phe Phe 1 5 10 15 <210> 31 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> MAGE-A3-alpha Chain Amino Acid Sequence <400> 31 Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val 1 5 10 15 Val Ser Ser Met Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser 20 25 30 Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg 35 40 45 Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu 50 55 60 Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile 65 70 75 80 Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn 85 90 95 Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys 100 105 110 Ala Leu Glu Val Arg Ser Ser Ala Ser Lys Ile Ile Phe Gly Ser Gly 115 120 125 Thr Arg Leu Ser Ile Arg Pro Asn Ile Gln Asn Pro Asp Pro Ala Val 130 135 140 Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe 145 150 155 160 Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp 165 170 175 Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe 180 185 190 Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys 195 200 205 Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro 210 215 220 Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu 225 230 235 240 Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg 245 250 255 Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg 260 265 270 Leu Trp Ser Ser Glx 275 <210> 32 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> MAGE-A3-beta chain amino acid sequence <400> 32 Met Leu Leu Leu Leu Leu Leu Leu Gly Pro Gly Ser Gly Leu Gly Ala 1 5 10 15 Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly Thr Ser 20 25 30 Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr Met Phe 35 40 45 Trp Tyr Arg Gin Phe Pro Lys Gin Ser Leu Met Leu Met Ala Thr Ser 50 55 60 Asn Glu Gly Ser Lys Ala Thr Tyr Gin Gin Gly Val Glu Lys Asp Lys 65 70 75 80 Phe Leu lie Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr...

Claims

1. A nucleic acid molecule comprising (a) A first nucleotide sequence encoding a recombinant T-cell receptor or its antigen-binding portion that specifically binds to a tyrosinase epitope, the epitope comprising an amino acid sequence as shown in SEQ ID NO:51, wherein the epitope is complexed with the HLA class I molecule HLA-C*05 allele; wherein the anti-tyrosinase TCR comprises an α chain and a β chain, wherein the α chain comprises a variable domain comprising α chain CDR1, α chain CDR2, and α chain CDR3; and wherein the β chain comprises a variable domain comprising β chain CDR1, β chain CDR2, and β chain CDR3; and wherein: (i) The amino acid sequence of the β chain CDR3 of the anti-tyrosinase TCR is shown in SEQ ID NO:10; (ii) The amino acid sequence of the β chain CDR2 of the anti-tyrosinase TCR is shown in SEQ ID NO:9; (iii) The amino acid sequence of the β chain CDR1 of the anti-tyrosinase TCR is shown in SEQ ID NO:8; (iv) The amino acid sequence of the α chain CDR3 of the anti-tyrosinase TCR is shown in SEQ ID NO:7; (v) The amino acid sequence of the α-chain CDR2 of the anti-tyrosinase TCR is shown in SEQ ID NO:6; and (vi) The amino acid sequence of the α-chain CDR1 of the anti-tyrosinase TCR is shown in SEQ ID NO:5; and (b) A second nucleotide sequence, wherein the second nucleotide sequence or the polypeptide encoded by the second nucleotide sequence inhibits the expression of endogenous TCR.

2. The nucleic acid molecule as described in claim 1, wherein the HLA class I molecule HLA-C*05 allele is selected from the HLA-C*05:01 allele, HLA-C*05:03 allele, HLA-C*05:04 allele, HLA-C*05:05 allele and HLA-C*05:06 allele.

3. The nucleic acid molecule as described in claim 1 or 2, wherein: (i) The α chain comprises the amino acid sequence as stated in SEQ ID NO:1; (ii) The β chain comprises the amino acid sequence as stated in SEQ ID NO:2; or (iii)(i) and (ii) both.

4. The nucleic acid molecule according to any one of claims 1 to 3, wherein the second nucleotide sequence (i) is one or more siRNAs that reduce the expression of endogenous TCRs, wherein the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of the endogenous TCR. (ii) Encode Cas9; or (iii)(i) and (ii) both.

5. The nucleic acid molecule of claim 4, wherein the one or more siRNAs comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NO:57-60.

6. A vector comprising a nucleic acid molecule as described in any one of claims 1 to 5.

7. The vector as described in claim 6 is a viral vector, a mammalian vector, or a bacterial vector.

8. The vector as described in claim 6 or 7, wherein it is a retroviral vector.

9. The vector according to any one of claims 6 to 8, wherein the vector is selected from the group consisting of: adenovirus vector, lentivirus vector, Sendai virus vector, baculovirus vector, Epstein-Barr virus vector, multivaccinia virus vector, vaccinia virus vector, herpes simplex virus vector, hybrid vector and adeno-associated virus vector.

10. The vector as described in any one of claims 6 to 9, wherein it is a lentiviral vector.

11. A cell comprising a nucleic acid molecule as claimed in any one of claims 1 to 5 or a vector as claimed in any one of claims 6 to 10.

12. The cell of claim 11, further expressing CD3.

13. The cell of claim 11 or 12, wherein the cell is a T cell.

14. The cell of claim 11 or 12, wherein the cell is a natural killer T cell or an ILC cell.

15. The cell of claim 11 or 12, wherein the cell is a natural killer cell.

16. Use of the cells as described in any one of claims 12 to 15 in the preparation of a medicament for treating melanoma in a subject in need.

17. The use as claimed in claim 16, wherein the melanoma is recurrent or refractory.

18. The use as claimed in claim 16, wherein the melanoma is a locally advanced cancer.

19. The use as claimed in claim 16, wherein the melanoma is an advanced cancer.

20. The use as claimed in claim 16, wherein the melanoma is a metastatic cancer.

21. The use as claimed in claim 16, wherein the cells are obtained from the subject.

22. The use as claimed in claim 16, wherein the cells are obtained from a donor other than the subject.

23. A method for engineered antigen-targeting cells, comprising transducing cells collected from a subject requiring T-cell therapy with a nucleic acid molecule as described in any one of claims 1 to 5 or a vector as described in any one of claims 6 to 10.

24. The method of claim 23, wherein the antigen-targeting cells further express CD3.

25. The method of claim 23, wherein the cell is a T cell.

26. The method of claim 23, wherein the cell is a natural killer cell.

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