T cell receptors and methods of use thereof

Abstract

To provide recombinant T cell receptors capable of binding a tyrosinase epitope, a MAGA-A1 epitope, a MART1 epitope, a MAGE-A3 epitope or an SSX2 epitope and nucleic acid molecules encoding the same.SOLUTION: In some embodiments, the nucleic acid molecules further comprise a second nucleotide sequence, where the second nucleotide sequence or the polypeptide encoded by the second nucleotide sequence inhibits the expression of an endogenous TCR. Other aspects of the disclosure are directed to vectors comprising the nucleic acid molecules and to cells comprising the recombinant TCRs, the nucleic acid molecules or the vectors. Still other aspects of the disclosure are directed to methods of using the same. In some embodiments, the methods comprise treating a cancer in a subject in need of cancer treatment.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 823,487, filed March 25, 2019, which is incorporated by reference in its entirety.

[0002] References to sequence listings submitted electronically via EFS-WEB The contents of the electronically submitted sequence listing (Name: 4285_008PC01_SeqListing_ST25.txt, Size: 69,638 bytes, and Creation Date: March 23, 2020) are incorporated by reference in their entirety into this specification.

[0003] The present disclosure provides recombinant T cell receptors ("TCRs") that specifically bind to a target human protein selected from the group consisting of tyrosinase, MAGE-A1, MART1, MAGE-A3, and SSX2, and uses thereof. [Background technology]

[0004] Immunotherapy has emerged as an important tool in the fight against various diseases, including cancer. T cell therapy is at the forefront of immunotherapy development, and adoptive transfer of antitumor T cells has been shown to induce clinical responses in cancer patients. Many T cell therapies target mutated tumor antigens, but the majority of neoantigens are not shared and are unique to each patient.

[0005] The number of potential non-mutated antigens outnumbers the mutated antigens by several orders of magnitude. Elucidation of T cell epitopes derived from shared antigens may facilitate the development of effective and safe adoptive T cell therapies that are readily accessible to larger cohorts of cancer patients. However, the sheer number of non-mutated antigens and the high polymorphism of HLA genes may hinder a comprehensive analysis of the specificity of anti-tumor T cell responses against non-mutated antigens.

[0006] The present disclosure provides novel epitopes for non-mutated antigens tyrosinase, MAGE-A1, MART1, MAGE-A3, and SXX2, and TCRs that can specifically bind to the epitopes. These novel epitopes are associated with specific HLA alleles. The use of these tumor-reactive HLA-restricted TCRs may broaden the scope of application of TCR gene therapy, especially in immuno-oncology. Summary of the Invention [Means for solving the problem]

[0007] Certain aspects of the present disclosure relate to nucleic acid molecules 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 (an "anti-tyrosinase TCR"), the anti-tyrosinase TCR cross-competes with a reference anti-tyrosinase TCR for binding to human tyrosinase, the reference anti-tyrosinase TCR comprising an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO:1 and the beta chain comprising the amino acid sequence set forth in SEQ ID NO:2; (b) the target human protein is MAGE-A1 (an "anti-MAGE-A1 TCR"), the anti-MAGE-A1 TCR cross-competes with a reference anti-MAGE-A1 TCR for binding to human MAGE-A1, the reference anti-MAGE-A1 TCR comprising an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO:11 and the beta chain comprising the amino acid sequence set forth in SEQ ID NO:12; and (c) the target human protein is MART1 (an "anti-MART1 TCR"), the anti-MART1 TCR cross-competes with a reference anti-MART1 TCR for binding to human MART1. (d) the target human protein is MAGE-A3 ("anti-MAGE-A3 TCR"), and the anti-MAGE-A3 TCR cross-competes with the reference anti-MAGE-A3 TCR for binding to human MAGE-A3, wherein the reference anti-MAGE-A3 TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 31 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO: 32; or (e) the target human protein is SSX2 ("anti-SSX2 TCR"), and the anti-SSX2 TCR cross-competes with the reference anti-SSX2 TCR for binding to human SSX2, wherein the reference anti-SSX2 TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 41 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO: 42.

[0008] Certain aspects of the present disclosure relate to nucleic acid molecules 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 (an "anti-tyrosinase TCR"), the anti-tyrosinase TCR binds to the same epitope as a reference anti-tyrosinase TCR or an overlapping epitope on human tyrosinase, the reference anti-tyrosinase TCR comprising an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO:1 and the beta chain comprising the amino acid sequence set forth in SEQ ID NO:2; (b) the target human protein is MAGE-A1 (an "anti-MAGE-A1 TCR"), the anti-MAGE-A1 TCR binds to the same epitope as a reference anti-MAGE-A1 TCR or an overlapping epitope on human MAGE-A1, the reference anti-MAGE-A1 TCR comprising an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO:11 and the beta chain comprising the amino acid sequence set forth in SEQ ID NO:12; and (c) the target human protein is MART1 (an "anti-MART1 TCR"), the anti-MART1 (d) the target human protein is MAGE-A3 ("anti-MAGE-A3 TCR") and the anti-MAGE-A3 TCR binds to the same epitope as the reference anti-MART1 TCR or an overlapping epitope of human MART1, the reference anti-MART1 TCR comprising an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO:21 and the beta chain comprising the amino acid sequence set forth in SEQ ID NO:22; (e) the target human protein is SSX2 ("anti-SSX2 TCR") and the anti-SSX2 TCR binds to the same epitope as the reference anti-SSX2 TCR or an overlapping epitope of human SSX2, the reference anti-SSX2 TCR comprising an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO:31 and the beta chain comprising the amino acid sequence set forth in SEQ ID NO:32; The TCR comprises an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO:41 and the beta chain comprising the amino acid sequence set forth in SEQ ID NO:42.

[0009] In some embodiments, (a) an anti-tyrosinase TCR binds to an epitope of tyrosinase consisting of the amino acid sequence set forth in SEQ ID NO:51, (b) an anti-MAGE-A1 TCR binds to an epitope of MAGE-A1 consisting of the amino acid sequence set forth in SEQ ID NO:52, (c) an anti-MART1 TCR binds to an epitope of MART1 consisting of the amino acid sequence set forth in SEQ ID NO:53, (d) an anti-MAGE-A3 TCR binds to an epitope of MAGE-A3 consisting of the amino acid sequence set forth in SEQ ID NO:54, or (e) an anti-SSX2 TCR binds to an epitope of SSX2 consisting of the amino acid sequence set forth in SEQ ID NO:55.

[0010] 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.

[0011] In some embodiments, (a) the target human protein is tyrosinase and the HLA class I molecule is selected from the HLA-C*05:01 allele, the HLA-C*05:03 allele, the HLA-C*05:04 allele, the HLA-C*05:05 allele, and the HLA-C*05:06 allele; (b) the target human protein is MAGE-A1 and the HLA class I molecule is selected from the HLA-B*07:02 allele, the HLA-B*07:03 allele, the HLA-B*07:04 allele, the HLA-B*07:05 allele, and the HLA-B*07:06 allele; and (c) the target human protein is MART1 and the HLA class I molecule is selected from the HLA-B*18:01 allele, the HLA-B*18 (d) the target human protein is MAGE-A3 and the HLA class I molecule is selected from the HLA-B*18:01 allele, the HLA-B*18:02 allele, the HLA-B*18:03 allele, the HLA-B*18:04 allele, and the HLA-B*18:05 allele; or (e) the target human protein is SSX2 and the HLA class I molecule is selected from the HLA-A*02:01 allele, the HLA-A*02:02 allele, the HLA-A*02:03 allele, the HLA-A*02:04 allele, and the HLA-A*02:05 allele.

[0012] 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.

[0013] In some embodiments, a recombinant TCR or antigen-binding portion thereof that specifically binds to a target human protein comprises an alpha chain and a beta chain, where the alpha chain comprises a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3, and the beta chain comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3, where (a) the target human protein is tyrosinase and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:7, (b) the target human protein is MAGE-A1 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:17, (c) the target human protein is MART1 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:27, (d) the target human protein is MAGE-A3 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:37, or (e) the target human protein is SSX2 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:47.

[0014] In some embodiments, (a) the target human protein is tyrosinase and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10, (b) the target human protein is MAGE-A1 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20, (c) the target human protein is MART1 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, (d) the target human protein is MAGE-A3 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40, or (e) the target human protein is SSX2 and the alpha beta CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0015] In some embodiments, a recombinant TCR or antigen-binding portion thereof that specifically binds to a target human protein comprises an alpha chain and a beta chain, where the alpha chain comprises a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3, and the beta chain comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3, where (a) the target human protein is tyrosinase and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10, (b) the target human protein is MAGE-A1 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20, (c) the target human protein is MART1 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, (d) the target human protein is MAGE-A3 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40, or (e) the target human protein is SSX2 and the alpha beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0016] In some embodiments, (a) the target human protein is tyrosinase and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:7; (b) the target human protein is MAGE-A1 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:17; (c) the target human protein is MART1 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:27; (d) the target human protein is MAGE-A3 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:37; or (e) the target human protein is SSX2 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:47.

[0017] In some embodiments, (a) the target human protein is tyrosinase and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:5; (b) the target human protein is MAGE-A1 and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:15; (c) the target human protein is MART1 and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:25; (d) the target human protein is MAGE-A3 and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:35; or (e) the target human protein is SSX2 and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:45.

[0018] In some embodiments, (a) the target human protein is tyrosinase and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (b) the target human protein is MAGE-A1 and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 18; (c) the target human protein is MART1 and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 28; (d) the target human protein is MAGE-A3 and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 38; or (e) the target human protein is SSX2 and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 48.

[0019] In some embodiments, (a) the target human protein is tyrosinase and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:6; (b) the target human protein is MAGE-A1 and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:16; (c) the target human protein is MART1 and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:26; (d) the target human protein is MAGE-A3 and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:36; or (e) the target human protein is SSX2 and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:46.

[0020] In some embodiments, (a) the target human protein is tyrosinase and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9; (b) the target human protein is MAGE-A1 and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19; (c) the target human protein is MART1 and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29; (d) the target human protein is MAGE-A3 and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 39; or (e) the target human protein is SSX2 and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49.

[0021] In some embodiments, (a) the target human protein is tyrosinase and the alpha chain variable domain comprises the amino acid sequence of the 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 the 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 the 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 the 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 the variable domain present in the amino acid sequence set forth in SEQ ID NO:41.

[0022] In some embodiments, (a) the target human protein is tyrosinase and the beta chain variable domain comprises the amino acid sequence of the 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 the 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 the 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 the 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 the 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, which constant region is distinct from the endogenous constant region of the alpha chain.

[0023] 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 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 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 the constant region present in the amino acid sequence set forth in SEQ ID NO:11; and (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 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 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 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 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 the constant region 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 alpha chain constant region comprises an amino acid sequence that contains at least one, at least two, at least three, at least four, or at least five 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 that contains at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO:11; and (c) the target human protein is MART1 and the alpha chain constant region comprises an amino acid sequence that contains at least one, at least two, at least three, at least four, or at least five 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 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 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 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 present in the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the beta chain further comprises a constant region, which is distinct from the endogenous constant region of the beta chain.

[0025] 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 the 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 the constant region present in the amino acid sequence set forth in SEQ ID NO:12; and (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 the 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 the 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 the constant region present in the amino acid sequence set forth in SEQ ID NO:42.

[0026] In some embodiments, (a) the target human protein is tyrosinase and the beta chain constant region comprises an amino acid sequence that contains at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the 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 that contains at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO:12; and (c) the target human protein is MART1 and the beta chain constant region comprises an amino acid sequence that contains at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the 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 that comprises at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the 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 that comprises at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO:42.

[0027] In some embodiments, (a) the target human protein is tyrosinase and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:1; (b) the target human protein is MAGE-A1 and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:11; (c) the target human protein is MART1 and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:21; (d) the target human protein is MAGE-A3 and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:31; or (e) the target human protein is SSX2 and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:41.

[0028] In some embodiments, (a) the target human protein is tyrosinase and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:2; (b) the target human protein is MAGE-A1 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:12; (c) the target human protein is MART1 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:22; (d) the target human protein is MAGE-A3 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:32; or (e) the target human protein is SSX2 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:42.

[0029] 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 Cas9.

[0030] In some embodiments, the recombinant TCR or antigen-binding portion thereof 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, comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five substitutions in the target sequence relative to the corresponding amino acid sequence of the endogenous TCR.

[0031] Certain aspects of the disclosure relate to vectors comprising the nucleic acid molecules 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.

[0032] 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.

[0033] Certain aspects of the present disclosure relate to recombinant T cell receptors (TCRs) or antigen-binding portions thereof ("recombinant TCRs") that specifically bind to a target human protein. wherein (a) the target human protein is tyrosinase (an "anti-tyrosinase TCR"), the anti-tyrosinase TCR cross-competes with a reference anti-tyrosinase TCR for binding to human tyrosinase, the reference anti-tyrosinase TCR comprising an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO:1 and the beta chain comprising the amino acid sequence set forth in SEQ ID NO:2; (b) the target human protein is MAGE-A1 (an "anti-MAGE-A1 TCR"), the anti-MAGE-A1 TCR cross-competes with a reference anti-MAGE-A1 TCR for binding to human MAGE-A1, the reference anti-MAGE-A1 TCR comprising an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO:11 and the beta chain comprising the amino acid sequence set forth in SEQ ID NO:12; and (c) the target human protein is MART1 (an "anti-MART1 TCR"), the anti-MART1 TCR cross-competes with a reference anti-MART1 TCR for binding to human MART1. (d) the target human protein is MAGE-A3 ("anti-MAGE-A3 TCR"), and the anti-MAGE-A3 TCR cross-competes with the reference anti-MAGE-A3 TCR for binding to human MAGE-A3, wherein the reference anti-MAGE-A3 TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 31 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO: 32; or (e) the target human protein is SSX2 ("anti-SSX2 TCR"), and the anti-SSX2 TCR cross-competes with the reference anti-SSX2 TCR for binding to human SSX2, wherein the reference anti-SSX2 TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 41 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO: 42.

[0034] Certain aspects of the present disclosure relate to recombinant T cell receptors (TCRs) or antigen-binding portions thereof ("recombinant TCRs") that specifically bind to a target human protein, where (a) the target human protein is tyrosinase (an "anti-tyrosinase TCR"), where the anti-tyrosinase TCR binds to the same epitope as a reference anti-tyrosinase TCR or an overlapping epitope on human tyrosinase, where the reference anti-tyrosinase TCR comprises an alpha chain and a beta chain, where the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:1 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:2, and (b) the target human protein is MAGE-A1 (an "anti-MAGE-A1 TCR"), where the anti-MAGE-A1 TCR binds to the same epitope as a reference anti-MAGE-A1 TCR or an overlapping epitope on human MAGE-A1, where the reference anti-MAGE-A1 TCR binds to the same epitope as a reference anti-MAGE-A1 TCR or an overlapping epitope on human MAGE-A1. (c) the target human protein is MART1 ("anti-MART1 TCR"), which binds to the same epitope as a reference anti-MART1 TCR or an overlapping epitope of human MART1, which reference anti-MART1 TCR comprises an alpha chain and a beta chain, which alpha chain comprises the amino acid sequence set forth in SEQ ID NO:21 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:22; (d) the target human protein is MAGE-A3 ("anti-MART1 TCR"), which binds to the same epitope as a reference anti-MART1 TCR or an overlapping epitope of human MART1, which reference anti-MART1 TCR comprises an alpha chain and a beta chain, which alpha chain comprises the amino acid sequence set forth in SEQ ID NO:21 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:22; or (e) the target human protein is SSX2 ("anti-SSX2 TCR"), wherein the anti-SSX2 TCR binds to the same epitope as the reference anti-MAGE-A3 TCR or an overlapping epitope on human MAGE-A3, and the reference anti-MAGE-A3 TCR comprises an alpha chain and a beta chain, and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:31 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:32; or (f) the target human protein is SSX2 ("anti-SSX2 TCR"), wherein the anti-SSX2 TCR binds to the same epitope as the reference anti-SSX2 TCR or an overlapping epitope on human SSX2, and the reference anti-SSX2 TCR comprises an alpha chain and a beta chain, and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:41 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:42.

[0035] In some embodiments, (a) an anti-tyrosinase TCR binds to an epitope of tyrosinase consisting of the amino acid sequence set forth in SEQ ID NO:51, (b) an anti-MAGE-A1 TCR binds to an epitope of MAGE-A1 consisting of the amino acid sequence set forth in SEQ ID NO:52, (c) an anti-MART1 TCR binds to an epitope of MART1 consisting of the amino acid sequence set forth in SEQ ID NO:53, (d) an anti-MAGE-A3 TCR binds to an epitope of MAGE-A3 consisting of the amino acid sequence set forth in SEQ ID NO:54, or (e) an anti-SSX2 TCR binds to an epitope of SSX2 consisting of the amino acid sequence set forth in SEQ ID NO:55.

[0036] 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.

[0037] In some embodiments, (a) the target human protein is tyrosinase and the HLA class I molecule is selected from the HLA-C*05:01 allele, the HLA-C*05:03 allele, the HLA-C*05:04 allele, the HLA-C*05:05 allele, and the HLA-C*05:06 allele; (b) the target human protein is MAGE-A1 and the HLA class I molecule is selected from the HLA-B*07:02 allele, the HLA-B*07:03 allele, the HLA-B*07:04 allele, the HLA-B*07:05 allele, and the HLA-B*07:06 allele; and (c) the target human protein is MART1 and the HLA class I molecule is selected from the HLA-B*18:01 allele, the HLA-B*18 (d) the target human protein is MAGE-A3 and the HLA class I molecule is selected from the HLA-B*18:01 allele, the HLA-B*18:02 allele, the HLA-B*18:03 allele, the HLA-B*18:04 allele, and the HLA-B*18:05 allele; or (e) the target human protein is SSX2 and the HLA class I molecule is selected from the HLA-A*02:01 allele, the HLA-A*02:02 allele, the HLA-A*02:03 allele, the HLA-A*02:04 allele, and the HLA-A*02:05 allele.

[0038] 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.

[0039] In some embodiments, a recombinant TCR or antigen-binding portion thereof that specifically binds to a target human protein comprises an alpha chain and a beta chain, where the alpha chain comprises a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3, and the beta chain comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3, where (a) the target human protein is tyrosinase and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:7, (b) the target human protein is MAGE-A1 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:17, (c) the target human protein is MART1 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:27, (d) the target human protein is MAGE-A3 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:37, or (e) the target human protein is SSX2 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:47.

[0040] In some embodiments, (a) the target human protein is tyrosinase and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10, (b) the target human protein is MAGE-A1 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20, (c) the target human protein is MART1 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, (d) the target human protein is MAGE-A3 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40, or (e) the target human protein is SSX2 and the alpha beta CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0041] In some embodiments, a recombinant TCR or antigen-binding portion thereof that specifically binds to a target human protein comprises an alpha chain and a beta chain, where the alpha chain comprises a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3, and the beta chain comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3, where (a) the target human protein is tyrosinase and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10, (b) the target human protein is MAGE-A1 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20, (c) the target human protein is MART1 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, (d) the target human protein is MAGE-A3 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40, or (e) the target human protein is SSX2 and the alpha beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0042] In some embodiments, (a) the target human protein is tyrosinase and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:7; (b) the target human protein is MAGE-A1 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:17; (c) the target human protein is MART1 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:27; (d) the target human protein is MAGE-A3 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:37; or (e) the target human protein is SSX2 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:47.

[0043] In some embodiments, (a) the target human protein is tyrosinase and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:5; (b) the target human protein is MAGE-A1 and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:15; (c) the target human protein is MART1 and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:25; (d) the target human protein is MAGE-A3 and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:35; or (e) the target human protein is SSX2 and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:45.

[0044] In some embodiments, (a) the target human protein is tyrosinase and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; (b) the target human protein is MAGE-A1 and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 18; (c) the target human protein is MART1 and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 28; (d) the target human protein is MAGE-A3 and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 38; or (e) the target human protein is SSX2 and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 48.

[0045] In some embodiments, (a) the target human protein is tyrosinase and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:6; (b) the target human protein is MAGE-A1 and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:16; (c) the target human protein is MART1 and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:26; (d) the target human protein is MAGE-A3 and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:36; or (e) the target human protein is SSX2 and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:46.

[0046] In some embodiments, (a) the target human protein is tyrosinase and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9; (b) the target human protein is MAGE-A1 and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19; (c) the target human protein is MART1 and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29; (d) the target human protein is MAGE-A3 and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 39; or (e) the target human protein is SSX2 and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49.

[0047] In some embodiments, (a) the target human protein is tyrosinase and the alpha chain variable domain comprises the amino acid sequence of the 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 the 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 the 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 the 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 the variable domain present in the amino acid sequence set forth in SEQ ID NO:41.

[0048] In some embodiments, (a) the target human protein is tyrosinase and the beta chain variable domain comprises the amino acid sequence of the 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 the 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 the 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 the 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 the variable domain present in the amino acid sequence set forth in SEQ ID NO:42.

[0049] 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 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 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 the constant region present in the amino acid sequence set forth in SEQ ID NO:11; and (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 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 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 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 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 the constant region present in the amino acid sequence set forth in SEQ ID NO:41.

[0050] 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 the 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 the constant region present in the amino acid sequence set forth in SEQ ID NO:12; and (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 the 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 the 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 the constant region present in the amino acid sequence set forth in SEQ ID NO:42.

[0051] In some embodiments, (a) the target human protein is tyrosinase and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:1; (b) the target human protein is MAGE-A1 and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:11; (c) the target human protein is MART1 and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:21; (d) the target human protein is MAGE-A3 and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:31; or (e) the target human protein is SSX2 and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:41.

[0052] In some embodiments, (a) the target human protein is tyrosinase and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:2; (b) the target human protein is MAGE-A1 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:12; (c) the target human protein is MART1 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:22; (d) the target human protein is MAGE-A3 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:32; or (e) the target human protein is SSX2 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:42.

[0053] Certain aspects of the disclosure relate to bispecific TCRs comprising a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain comprising a TCR or an antigen-binding portion thereof as disclosed herein, or a recombinant TCR as 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 an scFv. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are linked or joined by a covalent bond. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are linked by a peptide bond.

[0054] 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.

[0055] Certain aspects of the present disclosure relate to a method of treating cancer in a subject in need thereof comprising administering to the subject a cell as disclosed herein. In some embodiments, the cancer is selected from the group consisting of melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric 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, sarcoma of the bladder ... In some embodiments, the cancer is selected from the group consisting of cancer of the bladder, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of such cancers.

[0056] In some embodiments, the cancer is recurrent or refractory. In some embodiments, the cancer is locally advanced. In some embodiments, the cancer is advanced. In some embodiments, the cancer is metastatic.

[0057] In some embodiments, the cells are obtained from a subject. In some embodiments, the cells are obtained from a donor other than the subject. In some embodiments, the subject is pretreated prior to administration of the cells. In some embodiments, the pretreatment comprises administering to the subject a chemotherapy, a cytokine, a protein, a small molecule, or any combination thereof. In some embodiments, the pretreatment comprises administering an interleukin. In some embodiments, the pretreatment comprises administering IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof. In some embodiments, the pretreatment comprises administering a pretreatment agent selected from the group consisting of cyclophosphamide, fludarabine, vitamin C, an AKT inhibitor, ATRA, rapamycin, or any combination thereof. In some embodiments, the pretreatment comprises administering cyclophosphamide, fludarabine, or both.

[0058] Certain aspects of the present disclosure relate to methods for engineering antigen-targeted cells, comprising transducing cells collected from a subject in need of T cell therapy with a nucleic acid molecule disclosed herein or a vector disclosed herein. In some embodiments, the antigen-targeted cells further express CD3. In some embodiments, the cells are T cells or natural killer (NK) cells.

[0059] Certain aspects of the disclosure relate to an HLA class I molecule complexed to a peptide, the HLA class I molecule comprising an α1 domain, an α2 domain, an α3 domain, and a β2m, the peptide consisting 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 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.

[0060] 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.

[0061] In some embodiments, (a) the peptide comprises the amino acid sequence set forth in SEQ ID NO:51, and the HLA class I molecule is selected from 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 comprises the amino acid sequence set forth in SEQ ID NO:52, and the HLA class I molecule is selected from 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; and (c) the peptide comprises the amino acid sequence set forth in SEQ ID NO:53, and the HLA class I molecule is selected from an HLA-B*18:01 allele, an HLA- (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 HLA-B*18:01 allele, the HLA-B*18:02 allele, the HLA-B*18:03 allele, the HLA-B*18:04 allele, and the 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 HLA-A*02:01 allele, the HLA-A*02:02 allele, the HLA-A*02:03 allele, the HLA-A*02:04 allele, and the HLA-A*02:05 allele.

[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*05:01; (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.

[0063] In some embodiments, the HLA class I molecule is a monomer. In some embodiments, the HLA class I molecule is a dimer. In some embodiments, the HLA class I molecule is a trimer. In some embodiments, the HLA class I molecule is a tetramer. In some embodiments, the HLA class I molecule is a pentamer.

[0064] Certain aspects of the present disclosure relate to antigen-presenting cells (APCs) that comprise the HLA class I molecules disclosed herein. In some embodiments, the HLA class I molecules are expressed on the surface of the APCs.

[0065] Certain aspects of the disclosure relate to a method of enriching a target population of T cells obtained from a human subject, the method comprising contacting the T cells with an HLA class I molecule as disclosed herein or an APC as disclosed herein, wherein after the contacting, the enriched T cell population comprises a greater number of T cells capable of binding to the HLA class I molecule relative to the number of T cells capable of binding to the HLA class I molecule prior to the contacting.

[0066] Certain aspects of the disclosure relate to methods of enriching a target population of T cells obtained from a human subject, comprising contacting 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, and following said contacting, the enriched population of T cells comprises a greater number of T cells capable of targeting tumor cells relative to the number of T cells capable of targeting tumor cells prior to said contacting. In some embodiments, the T cells obtained from the human subject are tumor infiltrating lymphocytes (TILs).

[0067] Certain aspects of the present disclosure relate to methods of treating a tumor in a subject in need of such treatment, comprising administering to the subject the enriched T cells disclosed herein.

[0068] Certain aspects of the present disclosure relate to a method of enhancing cytotoxic T cell-mediated targeting of cancer cells in a subject suffering from 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.

[0069] 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.

[0070] Certain aspects of the present disclosure relate to a method of selecting T cells capable of targeting tumor cells, comprising contacting a population of in vitro isolated T cells with a peptide consisting 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 the drawings]

[0071] [Figure 1]A-D are graphical representations of C*05:01 / tyrosinase 460-468 multimer staining of melanoma TILs. TILs were stimulated once with C*05:01 artificial APCs pulsed with tyrosinase 460-468 peptide. Data are shown for C*05:01 / tyrosinase 460-468 (A-B) or control C*05:01 / HIV rev67-75 multimer (C-D) staining before stimulation (day 0, A and C) and 14 days after stimulation (day 14, B and D). The percentage of multimer+ cells among CD8+ T cells is shown. [Diagram 2] Bar graph showing functional evaluation of C*05:01 / tyrosinase 460-468 multimer positive melanoma TILs. IFN-γ production by C*05:01 positive TILs in a peptide-specific manner restricted to HLA-C*05:01. TILs were used as responder cells for IFN-γ ELISPOT assay. C*05:01 artificial APCs pulsed with the indicated peptides were used as stimulator cells. HIV rev67-75 peptide was used as control. Experiments were performed in triplicate and error bars indicate standard deviation (SD). *P<0.05. [Diagram 3] A-I are graphic representations of positive staining of Jurkat 76 / CD8 cells transduced with the C*05:01 / tyrosinase 460-468 TCR gene with the cognate multimer. Jurkat 76 / CD8 cells transduced with the C*05:01 / tyrosinase 460-468 TCR (B, E, and H) were stained with the C*05:01 / tyrosinase 460-468 multimer (B). In addition to non-TCR-transduced Jurkat 76 / CD8 (A, D, and G), C*05:01 / HIV rev67-75 multimers (D, E, and F), C*05:01 / non-exchanged multimers (G, H, and I), and C*07:02 / MAGE-A1289-297TCR (clone CL2; C, F, and I) were also used as controls. The percentages of multimer+CD8+ T cells are shown. [Figure 4]A-D are graphical representations of positive staining of human primary T cells transduced with the C*05:01 / tyrosinase 460-468 TCR gene (B and D) with the cognate multimer. Primary T cells transduced with the C*05:01 / tyrosinase 460-468 TCR were stained with C*05:01 / tyrosinase 460-468 (B) or C*05:01 / HIV rev67-75 control multimer (D). Non-transduced primary T cells were used as negative controls (A and C). The percentage of multimer+CD8+ T cells is shown. [Diagram 5] Bar graph showing that human primary T cells transduced with C*05:01 / tyrosinase 460-468 TCR gene strongly react with cognate peptides presented by target class I molecules. Primary T cells transduced with C*05:01 / tyrosinase 460-468 TCR gene or non-transduced primary T cells (x-axis) were used as responder cells for IFN-γ ELISPOT analysis. T2 cells transduced with HLA-C*05:01 (T2-C*05:01) were generated. T2 or T2-C05:01 cells pulsed with tyrosinase 460-468 or HIV rev67-75 peptide (control) were used as stimulator cells. Experiments were performed in triplicate and error bars indicate SD. ***P<0.001. [Figure 6] A, Graphical representation showing that primary T cells transduced with C*05:01 / tyrosinase 460-468 TCR gene recognize tumor cells. Primary T cells transduced with C*05:01 / tyrosinase 460-468 TCR gene or non-transduced primary T cells were used as responder cells for IFN-γ ELISPOT assay. Malme-3M, Me275, and MCF7 cells, non-transduced or transduced with HLA-C*05:01 or tyrosinase, were used as stimulator cells after treatment with 100 ng / ml IFNγ for 48 h, as indicated. Experiments were performed in triplicate, and error bars indicate SD. *P<0.05, **P<0.01, ***P<0.001. [Figure 7]A-D are graphical representations of the expression of endogenous or transduced tyrosinase genes in target cells analyzed via intracellular flow cytometry after staining with anti-tyrosinase mAb (open curves) and isotype control (closed curves). [Figure 8] A-D are graphical representations of the expression of ΔNGFR in target cells transduced with the full-length HLA-C*05:01 gene tagged with ΔNGFR (B and D). Surface expression of ΔNGFR in target cells transduced with the full-length HLA-C*05:01 gene tagged with ΔNGFR was analyzed by flow cytometry after staining with anti-NGFR mAb (open curve) and isotype control (closed curve). ΔNGFR alone was used as a control (A and C). [Figure 9] A-B are graphical representations of B*07:02 / MAGE-A1289-297 multimer staining of melanoma TILs. A shows B*07:02 / MAGE-A1289-297 multimer staining of TILs. B*07:02 / EBV EBNA3A379-387 (B) multimers were used as a negative control. The percentage of multimer+ cells among CD8+ T cells is shown. [Figure 10] Bar graph showing functional evaluation of B*07:02 / MAGE-A1289-297 multimer-positive melanoma TILs. IFN-γ production by TILs in a B*07:02 / MAGE-A1289-297-specific manner. TILs were used as responder cells for IFN-γ ELISPOT assay. B*07:02-artificial APCs pulsed with the indicated peptides were used as stimulator cells. HIV nef128-137 peptide was used as control. Experiments were performed in triplicate and error bars indicate standard deviation (SD). *P<0.05. [Figure 11]A-I are graphic representations of positive staining of Jurkat 76 / CD8 cells transduced with the B*07:02 / MAGE-A1289-297 TCR gene with the cognate multimer. Jurkat 76 / CD8 cells transduced with the B*07:02 / MAGE-A1289-297 TCR (B, E, and H) were stained with the B*07:02 / MAGE-A1289-297 multimer (B). In addition to non-TCR-transduced Jurkat 76 / CD8 (A, D, and G), B*07:02 / NY-ESO-160-72 multimers (D, E, and F), B*07:02 / non-exchanged multimers (G, H, and I), and B*07:02 / NY-ESO-160-72TCR (C, F, and I) were used as controls. The percentages of multimer+CD8+ cells are shown. [Figure 12] A-D are graphical representations of positive staining of human primary T cells transduced with the B*07:02 / MAGE-A1289-297 TCR gene (B and D) with the cognate multimer. Primary T cells transduced with B*07:02 / MAGE-A1289-297 TCR TCR were stained with B*07:02 / MAGE-A1289-297 (B) or B*07:02 / HIV nef128-137 control multimer (D). Non-transduced primary T cells were used as negative controls (A and C). The percentage of multimer+CD8+ T cells is shown. [Figure 13] Bar graph showing that human primary T cells transduced with B*07:02 / MAGE-A1289-297TCR gene strongly react with cognate peptides presented by target class I molecules. Primary T cells transduced with B*07:02 / MAGE-A1289-297TCR gene or non-transduced primary T cells (x-axis) were used as responder cells for IFN-γ ELISPOT analysis. T2 cells transduced with HLA-B*07:02 (T2-B*07:02) were generated. T2 or T2-B*07:02 cells pulsed with MAGE-A1289-297 or HIV nef128-137 peptide (control) were used as stimulator cells. Experiments were performed in triplicate and error bars indicate SD. **P<0.01. [Figure 14] A, Graphical representation showing that primary T cells transduced with B*07:02 / MAGE-A1289-297TCR gene recognize tumor cells. Primary T cells transduced with B*07:02 / MAGE-A1289-297TCR gene or non-transduced primary T cells were used as responder cells for IFN-γ ELISPOT analysis. Me275, SL-MEL-37, and SK-MEL-21 cells, non-transduced or transduced with HLA-B*07:02 or MAGE-A1, were used as stimulator cells as indicated (B). Experiments were performed in triplicate and error bars indicate SD. *P<0.05, **P<0.01. [Figure 15] A-D are graphical representations of the expression of MAGE-A1 derived from endogenous or transduced full-length genes. Expression of MAGE-A1 derived from endogenous or transduced full-length genes in target cells was analyzed via intracellular flow cytometry after staining with anti-MAGE-A1 mAb (open curves) and isotype control (closed curves). [Figure 16] A-D are graphical representations of the expression of ΔNGFR in target cells transduced with the full-length HLA-B*07:02 gene tagged with ΔNGFR (B and D). Surface expression of ΔNGFR in target cells transduced with the full-length HLA-B*07:02 gene tagged with ΔNGFR was analyzed by flow cytometry after staining with anti-NGFR mAb (open curve) and isotype control (closed curve). ΔNGFR alone was used as a control (A and C). [Figure 17]Bar graph showing the number of B*18:01 / MART1 T cells in melanoma TILs. TILs were used as responder cells in IFN-γ ELISPOT assay. B*18:01 artificial APCs pulsed with overlapping peptides to cover the entire protein of MART1 were used as stimulator cells. When stimulated with B*18:01 artificial APCs pulsed with overlapping peptides derived from MART1, TILs reacted positively against two adjacent peptides with the shared sequence 21YTTAEEAAGIGILTV35 (see also Table 5). Experiments were performed in triplicate, error bars indicate SD. *P<0.05, **P<0.001. [Figure 18] A-C are graphical representations of B*18:01 / MART125-33 multimer staining of melanoma TILs. B shows B*18:01 / MART125-33 multimer staining of TILs. B*18:01 / HIV gag161-170 (C) and B*18:01 / non-exchanged (A) multimers were used as negative controls. The percentage of multimer+ cells among CD8+ T cells is shown. [Figure 19] Bar graph showing functional evaluation of B*18:01 / MART125-33 multimer-positive melanoma TILs. IFN-γ production by TILs in a B*18:01 / MART125-33 specific manner. TILs were used as responder cells for IFN-γ ELISPOT assay. B*18:01 artificial APCs pulsed with the indicated peptides were used as stimulator cells. HIV gag161-170 peptide was used as control. Experiments were performed in triplicate and error bars indicate standard deviation (SD). ***P<0.001. [Figure 20]A-I are graphic representations of positive staining of Jurkat 76 / CD8 cells transduced with B*18:01 / MART125-33 TCR gene with cognate multimers. Jurkat 76 / CD8 cells transduced with B*18:01 / MART125-33 TCR (B, E, and H) were stained with B*18:01 / MART125-33 multimer (B). Besides Jurkat 76 / CD8 not transduced with TCR (A, D, and G), B*18:01 / MAGE-A3167-176 multimer (D, E, and F), B*18:01 / MAGE-A3167-176 TCR (C, F, and I), and B*18:01 / non-exchanged multimer (G, H, and I) were also used as controls. The percentage of multimer+CD8+ cells is shown. [Figure 21] A-D are graphic representations of positive staining of human primary T cells transduced with the B*18:01 / MART125-33 TCR gene (B and D) with cognate multimers. Primary T cells transduced with the B*18:01 / MART125-33 TCR were stained with B*18:01 / MART125-33 (B) or B*18:01 / HIV gag161-170 control multimer (D). Non-transduced primary T cells were used as negative controls (A and C). The percentage of multimer+CD8+ T cells is shown. [Figure 22] Bar graph showing that human primary T cells transduced with B*18:01 / MART125-33TCR gene strongly react with cognate peptides presented by target class I molecules. Primary T cells transduced with B*18:01 / MART125-33TCR gene or non-transduced primary T cells (x-axis) were used as responder cells for IFN-γ ELISPOT analysis. T2 cells transduced with HLA-B*18:01 (T2-B*18:01) were generated. T2 or T2-B*18:01 cells pulsed with MART125-33 or HIV gag161-170 peptide (control) were used as stimulator cells. Experiments were performed in triplicate and error bars indicate SD. **P<0.01. [Figure 23]A, Graphical representation showing that primary T cells transduced with B*18:01 / MART125-33TCR gene recognize tumor cells. Primary T cells transduced with B*18:01 / MART125-33TCR gene or non-transduced primary T cells were used as responder cells for IFN-γ ELISPOT analysis. Malme-3M, SL-MEL-28, and A375 cells, non-transduced or transduced with HLA-B*18:01 or MART1, were used as stimulator cells as indicated (B). Experiments were performed in triplicate and error bars indicate SD. *P<0.05, **P<0.01. [Figure 24] A-E are graphical representations of expression of MART1 from endogenous or transduced full-length genes. Expression of MART1 from endogenous or transduced full-length genes in target cells was analyzed via intracellular flow cytometry after staining with anti-MART1 mAb (open curves) and isotype control (closed curves). [Diagram 25] A-F are graphical representations of the expression of ΔNGFR in target cells transduced with the full-length HLA-B*18:01 gene tagged with ΔNGFR (B, D, and F). Surface expression of ΔNGFR in target cells transduced with the 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 (closed curve). ΔNGFR alone was used as a control (A, C, and E). [Figure 26] A-C are graphical representations of B*18:01 / MAGE-A3167-176 multimer staining of melanoma TILs. TILs were stimulated once with B*18:01 artificial APCs pulsed with MAGE-A3167-176 peptide. Data are shown for B*18:01 / MAGE-A3167-176 (A-B) or control B*18:01 / HIV gag161-170 multimer (C) staining before stimulation (day 0, A) and 14 days after stimulation (day 14, B-C). The percentage of multimer+ cells among CD8+ T cells is shown. [Figure 27] Bar graph showing functional evaluation of B*18:01 / MAGE-A3167-176 multimer-positive melanoma TILs. IFN-γ production by B*18:01-positive TILs in a peptide-specific manner restricted to HLA-B*18:01. TILs stimulated with B*18:01 artificial APCs pulsed with MAGE-A3167-176 peptides were used as responder cells for IFN-γ ELISPOT analysis. HLA-B*18:01-transduced T2 cells (T2-B*18:01) were generated. T2 or T2-B*18:01 cells pulsed with the indicated peptides were used as stimulator cells. HIV gag161-170 peptide was used as a control. Experiments were performed in triplicate and error bars indicate standard deviation (SD). **P<0.01, ***P<0.001. [Figure 28] A-I are graphic representations of positive staining of Jurkat 76 / CD8 cells transduced with B*18:01 / MAGE-A3167-176 TCR gene with cognate multimers. Jurkat 76 / CD8 cells transduced with B*18:01 / MAGE-A3167-176 TCR (B, E, and H) were stained with B*18:01 / MAGE-A3167-176 multimer (B). Besides Jurkat 76 / CD8 not transduced with TCR (A, D, and G), B*18:01 / MART125-33 multimer (D, E, and F), B*18:01 / non-exchanged multimer (G, H, and I), and B*18:01 / MART125-33 TCR (C, F, and I) were also used as controls. The percentage of multimer+CD8+ cells is shown. [Figure 29] A-D are graphical representations of positive staining of human primary T cells transduced with the B*18:01 / MAGE-A3167-176 TCR gene (B and D) with cognate multimers. Primary T cells transduced with the B*18:01 / MAGE-A3167-176 TCR were stained with B*18:01 / MAGE-A3167-176 (B) or B*18:01 / HIV gag161-170 control multimer (D). Non-transduced primary T cells were used as negative controls (A and C). The percentage of multimer+CD8+ T cells is shown. [Diagram 30] Bar graph showing that human primary T cells transduced with B*18:01 / MAGE-A3167-176TCR gene strongly react with cognate peptides presented by target class I molecules. Primary T cells transduced with B*18:01 / MAGE-A3167-176TCR gene or non-transduced primary T cells (x-axis) were used as responder cells for IFN-γ ELISPOT analysis. T2 cells pulsed with MAGE-A3167-176 or HIV gag161-170 peptide (control) were used as stimulator cells. Experiments were performed in triplicate and error bars indicate SD. **P<0.01. [Diagram 31] A, Graphical representation showing that primary T cells transduced with B*18:01 / MAGE-A3167-176TCR gene recognize tumor cells. Primary T cells transduced with B*18:01 / MAGE-A3167-176TCR gene or non-transduced primary T cells were used as responder cells for IFN-γ ELISPOT analysis. SK-MEL-28 and HEK293T cells, non-transduced or transduced with HLA-B*07:02 and / or MAGE-A1, were used as stimulator cells as indicated (B). Experiments were performed in triplicate and error bars indicate SD. *P<0.05, ***P<0.001. [Diagram 32] 1 is a graphical representation of MAGE-A3 expression from endogenous or transduced full-length genes. Expression of MAGE-A3 from endogenous or transduced full-length genes in target cells was assessed by Western blot analysis using anti-MAGE-A3 pAb. β-actin expression was used as a positive control. [Diagram 33]A-D are graphical representations of the expression of ΔNGFR in target cells transduced with the full-length HLA-B*18:01 gene tagged with ΔNGFR (B and D). Surface expression of ΔNGFR in target cells transduced with the 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 (closed curve). ΔNGFR alone was used as a control (A and C). [Diagram 34] (A-B) Graphical representation of A*02:01 / SSX241-49 multimer staining of melanoma TILs. (A) shows A*02:01 / SSX241-49 multimer staining of TILs. (B) A*02:01 / HTLV-1 tax11-19 multimer was used as a negative control. The percentage of multimer+ cells among CD8+ T cells is shown. [Diagram 35] Bar graph showing functional evaluation of A*02:01 / SSX241-49 multimer-positive melanoma TILs. IFN-γ production by TILs in an A*02:01 / SSX241-49-specific manner. TILs were used as responder cells for IFN-γ ELISPOT assay. T2 cells pulsed with the indicated peptides were used as stimulator cells. HTLV-1 tax11-19 peptide was used as control. Experiments were performed in triplicate and error bars indicate standard deviation (SD). **P<0.01. [Diagram 36]A-I are graphic representations of positive staining of Jurkat 76 / CD8 cells transduced with the A*02:01 / SSX241-49 TCR gene with the cognate multimer. Jurkat 76 / CD8 cells transduced with the A*02:01 / SSX241-49 TCR (B, E, and H) were stained with the A*02:01 / SSX241-49 multimer (B). In addition to non-TCR-transduced Jurkat 76 / CD8 (A, D, and G), A*02:01 / NY-ESO-1157-165 multimers (D, E, and F), A*02:01 / non-exchanged multimers (G, H, and I), and A*02:01 / NY-ESO-1157-165TCR (clone 1G4LY; C, F, and I) were used as controls. The percentages of multimer+CD8+ cells are shown. [Figure 37] A-D are graphical representations of positive staining of human primary T cells transduced with the A*02:01 / SSX241-49 TCR gene with the cognate multimer. Primary T cells transduced with the A*02:01 / SSX241-49 TCR (B and D) were stained with A*02:01 / SSX241-49 (B) or A*02:01 / HTLV-1 tax11-19 control multimer (D). Non-transduced primary T cells were used as negative controls (A and C). The percentage of multimer+CD8+ T cells is shown. [Figure 38] Bar graph showing that human primary T cells transduced with A*02:01 / SSX241-49TCR gene strongly react with cognate peptides presented by target class I molecules. Primary T cells transduced with A*02:01 / SSX241-49TCR gene or non-transduced primary T cells (x-axis) were used as responder cells in IFN-γ ELISPOT assay. T2 cells pulsed with SSX241-49 or HTLV-1 tax11-19 peptide (control) were used as stimulator cells. Experiments were performed in triplicate and error bars indicate SD. **P<0.01. [Figure 39]A, Graphical representation showing that primary T cells transduced with A*02:01 / SSX241-49TCR gene recognize tumor cells. Primary T cells transduced with A*02:01 / SSX241-49TCR gene or non-transduced primary T cells were used as responder cells in IFN-γ ELISPOT assay. SK-MEL-21, SL-MEL-37, and SK-MEL-28 cells, non-transduced or transduced with HLA-A*02:01 and / or SSX2, were used as stimulator cells as indicated (B). Experiments were performed in triplicate and error bars indicate SD. **P<0.01, ***P<0.001. [Diagram 40] 1 is a graphical representation of the expression of SSX2 from endogenous or transduced full-length genes. Expression of SSX2 from endogenous or transduced full-length genes in target cells was assessed by Western blot analysis using anti-SSX2 pAb. β-actin expression was used as a positive control. [Diagram 41] A-D are graphical representations of expression of HLA-A2 derived from endogenous or transduced full-length HLA-A*02:01 genes in target cells. Surface expression of HLA-A2 derived from endogenous or transduced full-length HLA-A*02:01 in target cells was analyzed via flow cytometry after staining with anti-HLA-A2 mAb (open curve) and isotype control (closed curve). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] The present disclosure relates to a TCR or antigen-binding portion thereof that specifically binds to an epitope on a target human protein selected from the group consisting of tyrosinase, MAGE-A1, MART1, MAGE-A3, and SSX2, a nucleic acid molecule encoding the same, and a cell comprising the TCR or nucleic acid molecule. Some aspects of the disclosure relate to a method of treating cancer in a subject in need thereof, comprising administering to the subject the cell. Other aspects of the disclosure relate to an HLA class I molecule complexed to a peptide comprising a tyrosinase, MAGE-A1, MART1, MAGE-A3, or SSX2 epitope.

[0073] I. Terminology In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise stated herein, each of the following terms shall have the meaning indicated below. Additional definitions are set forth throughout this application.

[0074] It should be noted that the term "a" or "an" entity refers to one or more of that entity, for example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0075] Furthermore, "and / or" as used herein shall be construed as a specific disclosure of each of two particular features or components, with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0076] The term "about" is used herein to mean approximately, roughly, roughly, or within a range thereof. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value with a variance (increase or decrease (higher or lower)) of 10%.

[0077] Whenever an embodiment is described herein with the word "comprising," it is to be understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. See, for example, 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 Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0079] Units, prefixes, and symbols are shown in their International System of Units (SI) accepted format. Numeric ranges are inclusive of the numbers defining the range. Nucleotide sequences are written left to right in 5' to 3' orientation unless otherwise indicated. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not intended to limit the various aspects of the disclosure, which may be had by reference to the specification in its entirety. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0080] "Administering" refers to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration" as used herein means a method of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered by a route other than parenteral, for example, orally. Other non-parenteral routes include topical, epithelial, or mucosal routes of administration, such as intranasally, intravaginally, rectally, sublingually, or topically, and administration can be, for example, once, multiple times, and / or over one or more extended periods of time.

[0081] The term "T cell receptor" (TCR) as used herein refers to a heteromeric cell surface receptor that can specifically interact 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 their antigen-binding portions, chimeric TCRs, TCR fusion constructs, and synthetic TCRs. In humans, TCRs are expressed on the surface of T cells and 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 (MHC; also referred to herein as complexed with HLA molecules, e.g., HLA class 1 molecules). TCRs recognize and bind antigen:HLA complexes and recruit CD3 (expressed by T cells) to activate the TCR. Activated TCRs initiate downstream signaling and immune responses, including the destruction of EPCs.

[0082] Generally, a TCR may comprise two chains, an alpha chain and a beta chain (or less commonly a gamma chain and a delta chain), 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 the antigen. The constant region is located proximal to the cell membrane. The TCR may further comprise a transmembrane region and a short cytoplasmic tail. As used herein, the term "constant region" encompasses the transmembrane region and the cytoplasmic tail, if present, as well as the conventional "constant region."

[0083] Variable domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), flanked by more conserved regions, termed framework regions (FRs). Each alpha and beta chain variable domain contains three CDRs and four FRs (FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4). Each variable domain contains a binding domain that interacts with antigen. All three CDRs of each chain are involved in antigen binding, although CDR3 is thought to be the major antigen binding region. CDR1 also interacts with antigen, while CD2 is thought to primarily recognize the HLA complex.

[0084] Unless expressly stated and unless the context dictates otherwise, the term "TCR" also includes antigen-binding fragments or portions of any TCR disclosed herein, including monovalent and bivalent fragments or portions, and single chain TCRs. The term "TCR" is not limited to naturally occurring TCRs bound to the surface of T cells. 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 does not include a cell membrane (e.g., an isolated TCR or a soluble TCR).

[0085] An "antigen binding molecule," "part of a TCR," or "TCR fragment" refers to any portion of a TCR that is less than the entire TCR. An antigen binding molecule may include the complementarity determining regions (CDRs) of an antigen.

[0086] An "antigen" refers to any molecule, e.g., a peptide, that can elicit an immune response or be bound by a TCR. An "epitope," as used herein, refers to a portion of a polypeptide that can elicit an immune response or be bound by a TCR. The immune response can include antibody production, or activation of specific immunologically competent cells, or both. Those skilled in the art will readily appreciate that virtually any macromolecule, including any protein or peptide, can function as an antigen. The antigen and / or epitope can be endogenously expressed, i.e., expressed by genomic DNA, or recombinantly expressed. The antigen and / or epitope can be specific to a particular tissue, such as a cancer cell, or it can be broadly expressed. Additionally, fragments of larger molecules can function as antigens. In one embodiment, the antigen is a tumor antigen. The epitope can be present on a longer polypeptide (e.g., a protein), or the epitope can be present 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).

[0087] As used herein, "tyrosinase" or "TYR" (UniProtKB-P14679) refers to a copper-containing oxidase that functions in the formation of pigments such as melanin and other polyphenolic compounds. Tyrosinase catalyzes the initial and rate-limiting step in the cascade of reactions that lead to melanin production 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 of the human body. The canonical tyrosinase amino acid sequence (SEQ ID NO: 89) is shown in Table 1.

[0088] As used herein, "melanoma associated antigen 1" or "MAGE-A1" (UniProtKB-P43355) refers to a tumor antigen expressed in various types of cancer, including melanoma, head and neck squamous cell carcinoma, lung cancer, and breast cancer. MAGE-A1 is not expressed in normal tissues other than the testis. MAGE-A1 is thought to be involved in transcriptional regulation through interaction with SNW1 and recruits the histone deactivation enzyme HDAC1. MAGE-A1 also inhibits transactivation of the Notch intracellular domain (NICD) and is thought to potentially play a role in aspects of embryonic development and tumor transformation, or tumor progression. The MAGE-A1 amino acid sequence (SEQ ID NO: 90) is shown in Table 1.

[0089] As used herein, "MART1", "MART-1", or "Melanoma antigen recognized by T cells 1" (UniProtKB-Q16655) refers to an antigen that is 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 important for the formation of stage II melanosomes. MART1 is expressed in melanoma cells, maranocytes, and retina. The MART1 amino acid sequence (SEQ ID NO: 91) is shown in Table 1.

[0090] As used herein, "MAGE-A3" or "melanoma associated antigen 3" (UniProtKB-P43357) refers to an antigen believed to enhance the ubiquitin ligase activity of RING-type zinc finger-containing E3 ubiquitin protein ligases. MAGE-A3 may also act to enhance the ubiquitin ligase activity of TRIM28 and stimulate ubiquitination of p53 / TP53 by TRIM28. MAGE-A3 is also believed to act through the recruitment and / or stabilization of Ubl-conjugating enzymes (E2) in E3:substrate complexes. MAGE-A3 may also be involved 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 various types of cancer, including melanoma, head and neck squamous cell carcinoma, lung cancer, and breast cancer. MAGE-A3 is not expressed in normal tissues other than testis and placenta. The amino acid sequence of MAGE-A3 (SEQ ID NO: 92) is shown in Table 1.

[0091] As used herein, "SSX2" or "protein SSX2" (UniProtKB-Q16385) is an antigen expressed in rhabdomyosarcoma and fibrosarcoma cell lines. SSX2 is also expressed at high levels in the testis and at lower levels in the thyroid. The function of SSX2 is unclear, although it has been speculated that SSX2 may act as a modulator of transcription. The SSX2 amino acid sequence (SEQ ID NO: 93) is shown in Table 1. [Table 1-1] [Table 1-2]

[0092] The term "HLA" as used herein refers to human leukocyte antigens. HLA genes code for human major histocompatibility complex (MHC) proteins. MHC proteins are expressed on the surface of cells and are involved in activating immune responses. HLA class I genes code for MHC class I molecules that are expressed on the surface of cells in complexes with peptide fragments of self or non-self proteins (antigens). T cells expressing TCR and CD3 recognize antigen:MHC class I complexes and initiate an immune response to target and destroy antigen-presenting cells that present non-self proteins.

[0093] 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 an MHC class I molecule. Thus, "HLA class I molecule" and "MHC class I molecule" are used interchangeably herein.

[0094] MHC class I molecules contain two protein chains: an alpha chain and a β2-microglobulin (β2m) chain. Human β2m is encoded by the B2M gene. The amino acid sequence of β2m is shown 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 in the 6p21.3 region of the short arm of human chromosome 6 and contains more than 220 genes of diverse functions. HLA genes are highly diverse, with more than 20,000 HLA alleles and associated alleles, including more than 15,000 HLA class I alleles known in the art, encoding thousands of HLA proteins, including more than 10,000 HLA class I proteins (see, for example, hla.alleles.org (last accessed February 27, 2019)). In the HLA complex, there are at least three genes that code for MHC class I alpha chain proteins: HLA-A, HLA-B, and HLA-C. In addition, HLA-E, HLA-F, and HLA-G code for proteins that associate with MHC class I molecules. [Table 2]

[0095] The term "autologous" refers to any material derived from the same individual that is subsequently reintroduced. For example, autologous T cell therapy involves administering to a subject T cells isolated from the same subject. The term "allogeneic" refers to any material derived from one individual that is then introduced into another individual of the same species. For example, allogeneic T cell transplantation involves administering to a subject T cells obtained from a donor other than the subject.

[0096] "Cancer" refers to a broad group of diseases characterized by uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors that invade adjacent tissues and may metastasize to distant parts of the body via 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 invention 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 invention are directed to treating a variety of cancers, including, for example, bone cancer, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, cutaneous or intraocular malignant melanoma, pancreatic cancer, skin cancer, head and neck cancer, cutaneous 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), cancer of the esophagus, 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, It may be used to reduce tumor size in tumors resulting from carcinoma, soft tissue sarcoma, 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), childhood solid tumors, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of the above cancers. Certain cancers may respond to chemotherapy or radiation therapy, or the cancers may be refractory. Refractory cancer refers to cancer that cannot be corrected by surgical intervention, the cancer does not respond initially to chemotherapy or radiation therapy, or the cancer becomes unresponsive over time.

[0097] As used herein, "anti-tumor effect" refers to a biological effect that can be manifested 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 survival or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with tumors.Anti-tumor effect can also refer to the prevention of tumor development, for example, vaccination.

[0098] The term "progression-free survival", sometimes abbreviated as PFS, as used herein, refers to the time from the date of treatment to the date of disease progression or death from any cause according to the revised IWG Response Criteria for Malignant Lymphoma.

[0099] "Progressive disease", which may be abbreviated as "disease progression" or PD, as used herein, refers to the worsening of one or more symptoms associated with a particular disease. For example, disease progression in a subject suffering from cancer may include an increase in the number or size of one or more malignant lesions, tumor metastasis, and death.

[0100] "Duration of response," which may be abbreviated as DOR, as used herein, refers to the time from a subject's first objective response to the date of confirmed disease progression or death according to the revised IWG Response Criteria for Malignant Lymphoma.

[0101] The term "overall survival," which may be abbreviated as OS, is defined as the time from the date of treatment to the date of death.

[0102] As used herein, a "cytokine" is a non-antibody protein released by one cell in response to contact with a specific antigen, which interacts with a second cell to mediate a response in the second cell. Cytokines may be expressed endogenously by cells or administered to a subject. Cytokines may be released from immune cells, such as macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines may induce a variety of responses in recipient cells. Cytokines may include homeostatic cytokines, chemokines, proinflammatory cytokines, effectors, and acute phase proteins. For example, homeostatic cytokines, such as interleukin (IL) 7 and IL-15, may promote survival and proliferation of immune cells, and proinflammatory cytokines may promote inflammatory responses. 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) gamma. Examples of proinflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, 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 placenta 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).

[0103] A "chemokine" is a type of cytokine that mediates chemotaxis, or directional 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 chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1 alpha, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC or CCL17).

[0104] 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 chemoattractant 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), oncoprotein (protein I), and IL-1 protein (protein II). These include statin M (OSM), CD154, lymphotoxin (LT) beta, 4-1BB ligand (4-1BBL), 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).

[0105] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of disease or promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods of the disease, or prevention of impairment or disability due to the affliction of the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled physician, for example, by assaying the activity of the agent in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0106] The term "lymphocyte" as used herein includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell-toxic) lymphocyte and are a major component of the innate immune system. NK cells reject tumor and virus-infected cells. It functions through the process of apoptosis or programmed cell death. They were 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, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells: helper T cells (e.g., CD4+ cells), cytotoxic T cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytotoxic T cells, CD8+ T cells, or killer T cells), memory T cells ((i) stem memory T cells such as naive cells, SCM The cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, exhibiting many functional attributes characteristic of memory cells); (ii) central memory T CMThe cells express L-selectin and CCR7 and secrete IL-2, but not IFNγ or IL-4, and (iii) effector memory T EM These cells are: T cells (which do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Treg, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. On the other hand, B cells play a major role in humoral immunity (involving the participation of antibodies). B cells make antibodies and antigens, act as antigen-presenting cells (APC), and turn into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, hence the name.

[0107] The term "genetically engineered" or "engineered" refers to a method of modifying the genome of a cell, including, but not limited to, deleting a coding or non-coding region or a portion thereof, or inserting a coding region or a portion thereof. In some embodiments, the modified cell is a lymphocyte, e.g., a T cell or a modified cell expressing CD3, which can be obtained from either a patient or a donor. The cell can be modified to express an exogenous construct, such as, for example, a T cell receptor (TCR) as disclosed herein, which is integrated into the genome of the cell. In some embodiments, the cell is modified to express CD3.

[0108] "Immune response" refers to the actions 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 the soluble macromolecules produced by any of these cells or the liver, including antibodies (Abs), cytokines, and complement, that result in the elimination of invading pathogens from the vertebrate body and / or the selective targeting, binding, damage, and destruction of pathogen-infected cells or tissues, cancer cells or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0109] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of suffering from or recurring from a disease by a method that involves inducing, enhancing, suppressing, or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy includes adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation.

[0110] The cells used in the immunotherapy described herein may be derived from sources known in the art. For example, T cells may be differentiated in vitro from a hematopoietic stem cell population, or the T cells may be obtained from a subject. T cells may be obtained, for example, from 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. Additionally, T cells may be derived from one or more T cell lines available in the art. T cells may also be obtained from a unit of blood collected from a subject using a number of techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. Additional methods of isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety. Immunotherapy may also include administering modified cells to a subject, where the modified cells express CD3 and TCR as disclosed herein. In some embodiments, the modified cells are not T cells.

[0111] As used herein, a "patient" includes any human suffering from cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0112] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, as well as longer chains, of which there are many types, commonly referred to in the art as proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0113] As used herein, "stimulation" refers to a primary response induced by the binding of a stimulatory molecule to its cognate ligand, which mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell, e.g., a 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, etc.), specifically binds to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. 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.

[0114] The terms "treatment" and "pretreatment" are used interchangeably herein to refer to preparing a patient in need of T cell therapy for an appropriate condition. Treatment as used herein includes, but is not limited to, reducing the number of endogenous lymphocytes, removing cytokine sinks, increasing serum levels of one or more homeostatic cytokines or proinflammatory factors, enhancing effector function of T cells administered after treatment, enhancing activation and / or availability of antigen presenting cells, or any combination thereof prior to T cell therapy. In one embodiment, "treatment" comprises increasing the 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-inducible protein 10 (IP-10), interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), placenta 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, "treatment" comprises increasing the serum levels of IL-7, IL-15, IP-10, MCP-1, PLGF, CRP, or any combination thereof.

[0115] "Treatment" or "treating" of a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, mitigating, ameliorating, inhibiting, slowing, or preventing the onset, progression, development, severity of symptoms, or recurrence of a symptom, complication or condition, or biochemical manifestations associated with a disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission.

[0116] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any listed or enumerated components.

[0117] The term "about" or "essentially comprising" refers to a value or composition that is within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially comprising" can mean within or more than one standard deviation per practice in the art. Alternatively, "about" or "essentially comprising" can mean a range of up to 10% (i.e., ±10%). For example, about 3 mg can include any number between 2.7 mg and 3.3 mg (at 10%). Furthermore, particularly with respect to biological systems or processes, the term can mean a value up to an order of magnitude or up to 5 times. When a particular value or composition is provided in this application and claims, unless otherwise indicated, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range of that particular value or composition.

[0118] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified.

[0119] Various aspects of the invention are described in further detail in the following subsections.

[0120] II. Compositions of the Disclosure The present disclosure relates to a T cell receptor (TCR) or an antigen-binding portion thereof that specifically binds to an epitope on a target human protein selected from the group consisting of tyrosinase, MAGE-A1, MART1, MAGE-A3, and SSX2, a nucleic acid molecule encoding the same, and a cell comprising the TCR or nucleic acid molecule. Some aspects of the disclosure relate to a method of treating cancer in a subject in need thereof comprising administering to the subject a cell comprising a TCR as described herein. Other aspects of the disclosure relate to an HLA class I molecule complexed to an epitope of tyrosinase, MAGE-A1, MART1, MAGE-A3, or SSX2 to which the TCR binds, and a peptide comprising an epitope of tyrosinase, MAGE-A1, MART1, MAGE-A3, or SSX2.

[0121] T cell receptors, or TCRs, are molecules found on the surface of T cells, or T lymphocytes, that are involved in recognizing fragments of antigens as peptides bound to major histocompatibility complex (MHC) molecules. The binding between TCRs and antigenic peptides is relatively low affinity and degenerate: that is, many TCRs recognize the same antigenic peptide and many antigenic peptides are recognized by the same TCR.

[0122] The 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 alpha (α) and beta (β) chains (encoded by TRA and TRB, respectively), whereas in 5% of T cells, the TCR is composed of gamma and delta (γ / δ) chains (encoded by TRG and TRD, respectively). This ratio changes during ontogeny and in pathological conditions (e.g., leukemia). It also differs between species. Orthologues of the four loci have been mapped in different species. Each locus can generate different polypeptides with constant and variable regions.

[0123] When the TCR engages an antigenic peptide and MHC (peptide / MHC), the T lymphocyte is activated through signal transduction, a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.

[0124] II.A. Nucleic acid molecules Certain aspects of the present disclosure relate to a nucleic acid molecule comprising: (i) a first nucleotide sequence encoding a recombinant TCR or antigen-binding portion thereof ("epitope-specific TCR") 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, where the second nucleotide sequence or the 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 synthetic. In still 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 with a reference TCR for binding to the target human protein. In some embodiments, the TCR binds to the same epitope or an overlapping epitope of the target human protein as the reference TCR.

[0125] In some embodiments, the reference TCR comprises an alpha chain and a beta chain, where the alpha chain comprises complementarity determining region 1 (CDR1), CDR2, and CDR3, and the beta chain comprises CDR1, CDR2, and CDR3, where the reference TCR comprises an alpha chain CDR3 amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 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 NOs: 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.

[0126] 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 NOs: 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 NOs: 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 the amino acid sequence set forth in SEQ ID NO: 1, 11, 21, 31, or 41, and the beta chain comprises the amino acid sequence set forth in SEQ ID NO: 2, 12, 22, 32, or 42. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11]

[0127] II.A.1. Epitope-specific TCR Certain aspects of the present disclosure relate to epitope-specific TCRs. In some embodiments, the epitope-specific TCR is encoded by a first nucleotide sequence described herein. In some embodiments, for example, the epitope-specific TCR 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, for example, the epitope-specific TCR encoded by the first nucleotide sequence comprises an alpha chain and a beta chain, where the alpha chain comprises a variable domain comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3, and the beta chain comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3.

[0128] II.A.1.a. Anti-tyrosinase TCR In some embodiments, an epitope-specific TCR, e.g., an epitope-specific TCR encoded by a first nucleotide sequence, specifically binds to an epitope on human tyrosinase (an "anti-tyrosinase TCR"), and the anti-tyrosinase TCR comprises an alpha chain CDR3 that comprises the amino acid sequence set forth in SEQ ID NO:7 (CLVGDVEGSQGNLIF). In some embodiments, the anti-tyrosinase TCR comprises a beta chain CDR3 that comprises the amino acid sequence set forth in SEQ ID NO:10 (CASSHHSGGIYNEQFF). In some embodiments, for example, an anti-tyrosinase TCR encoded by a first nucleotide sequence comprises an alpha chain CDR1, and the alpha chain CDR1 of the anti-tyrosinase TCR comprises the amino acid sequence set forth in SEQ ID NO:5 (NIATNDY). In some embodiments, the anti-tyrosinase TCR, e.g., the epitope-specific TCR encoded by the first nucleotide sequence, comprises a beta chain CDR1, and the beta chain CDR1 of the anti-tyrosinase TCR comprises the amino acid sequence (MNHEY) shown in SEQ ID NO:8.

[0129] In some embodiments, for example, an anti-tyrosinase TCR encoded by a first nucleotide sequence comprises an alpha chain CDR2, which comprises the amino acid sequence (GYKTK) set forth in SEQ ID NO: 6. In some embodiments, for example, an anti-tyrosinase TCR encoded by a first nucleotide sequence comprises a beta chain CDR2, which comprises the amino acid sequence (SVGAGI) set forth in SEQ ID NO: 9.

[0130] In certain embodiments, for example, the anti-tyrosinase TCR encoded by the first nucleotide sequence 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.

[0131] In some embodiments, the non-CDR regions of the alpha and / or beta chains of an epitope-specific TCR, such as an anti-tyrosinase TCR, are further modified, e.g., by substitution or mutation of one, two, three, four, five, or six amino acids, such that the alpha and / or beta chain is not naturally occurring. In some embodiments, the substitutions or mutations may improve the TCRs described herein in various aspects, e.g., binding affinity, binding specificity, stability, viscosity, or any combination thereof.

[0132] In some embodiments, for example, an anti-tyrosinase TCR encoded by a 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, for example, an anti-tyrosinase TCR encoded by a 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, and the anti-tyrosinase TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, for example, the anti-tyrosinase TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain present in the alpha chain amino acid sequence shown in SEQ ID NO:1.

[0133] In some embodiments, for example, an anti-tyrosinase TCR encoded by a 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, for example, an anti-tyrosinase TCR encoded by a 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, and the anti-tyrosinase TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, for example, the anti-tyrosinase TCR encoded by the first nucleotide sequence comprises a beta chain variable domain present in the amino acid sequence shown in SEQ ID NO:2.

[0134] In some embodiments, for example, 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, for example, the anti-tyrosinase TCR encoded by the first nucleotide sequence comprises an alpha chain constant 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 constant region of the alpha chain amino acid sequence set forth in SEQ ID NO:1. In some embodiments, for example, an anti-tyrosinase TCR encoded by a 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, and the anti-tyrosinase TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, for example, an anti-tyrosinase TCR encoded by a 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, for example, an anti-tyrosinase TCR encoded by a first nucleotide sequence further comprises an alpha constant region that is distinct 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 one, at least two, at least three, at least four, or at least five 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.

[0135] In some embodiments, for example, an anti-tyrosinase TCR encoded by a 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, for example, an anti-tyrosinase TCR encoded by a 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, and the anti-tyrosinase TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, for example, an anti-tyrosinase TCR encoded by a first nucleotide sequence comprises a beta chain constant region present in the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, for example, an anti-tyrosinase TCR encoded by a first nucleotide sequence further comprises a beta constant region that is distinct 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 one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the constant region of the beta chain amino acid sequence set forth in SEQ ID NO:2.

[0136] In certain embodiments, for example, an anti-tyrosinase TCR encoded by a 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, for example, an anti-tyrosinase TCR encoded by a 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, and the anti-tyrosinase TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, for example, the anti-tyrosinase TCR encoded by the first nucleotide sequence comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO:1.

[0137] In certain embodiments, for example, an anti-tyrosinase TCR encoded by a 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, for example, an anti-tyrosinase TCR encoded by a 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, where the anti-tyrosinase TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, for example, the anti-tyrosinase TCR encoded by the first nucleotide sequence comprises a beta chain comprising the amino acid sequence shown in SEQ ID NO:2.

[0138] In some embodiments, for example, an anti-tyrosinase TCR encoded by a first nucleotide sequence comprises an alpha chain constant region, a beta chain constant region, or both, where the alpha chain constant region, the beta chain constant region, or both, comprises an amino acid sequence that has at least one, at least two, at least three, at least four, or at least five substitutions in the target sequence relative to the corresponding amino acid sequence of an endogenous TCR. In some embodiments, for example, an anti-tyrosinase TCR encoded by a first nucleotide sequence comprises an alpha chain and a beta chain, where the alpha chain comprises a constant region and the beta chain comprises a constant region, where (i) the alpha chain constant region comprises an amino acid sequence that has at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the alpha chain that comprises the amino acid sequence set forth in SEQ ID NO:1, and (ii) the beta chain constant region comprises an amino acid sequence that has at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the beta chain that comprises the amino acid sequence set forth in SEQ ID NO:2.

[0139] In some embodiments, for example, an anti-tyrosinase TCR encoded by a first nucleotide sequence cross-competes with a reference TCR for binding to human tyrosinase. In some embodiments, the anti-tyrosinase TCR binds to the same epitope of human tyrosinase as the reference TCR or an overlapping epitope. In some embodiments, the reference TCR comprises an alpha chain and a beta chain, and the alpha chain of the reference TCR comprises the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the beta chain of the reference TCR comprises the amino acid sequence set forth in SEQ ID NO:2.

[0140] II.A.1.b. Anti-MAGE-A1 TCR In some embodiments, the epitope-specific TCR, e.g., the epitope-specific TCR encoded by the first nucleotide sequence, specifically binds to an epitope on human MAGE-A1 (an "anti-MAGE-A1 TCR"), and the anti-MAGE-A1 TCR comprises an alpha chain CDR3 that comprises the amino acid sequence (CALSESYSGAGSYQLTF) set forth in SEQ ID NO: 17. In some embodiments, the anti-MAGE-A1 TCR comprises a beta chain CDR3 that comprises the amino acid sequence (CASSLASGSNQPQHF) set forth in SEQ ID NO: 20.

[0141] In some embodiments, for example, the anti-MAGE-A1 TCR encoded by the first nucleotide sequence comprises an alpha chain CDR1, which comprises the amino acid sequence (TRDTTYYL) set forth in SEQ ID NO: 15. In some embodiments, for example, the anti-MAGE-A1 TCR encoded by the first nucleotide sequence comprises a beta chain CDR1, which comprises the amino acid sequence (SEHNR) set forth in SEQ ID NO: 18.

[0142] In some embodiments, for example, the anti-MAGE-A1 TCR encoded by the first nucleotide sequence comprises an alpha chain CDR2, which comprises the amino acid sequence (RNSFDEQN) set forth in SEQ ID NO: 16. In some embodiments, for example, the anti-MAGE-A1 TCR encoded by the first nucleotide sequence comprises a beta chain CDR2, which comprises the amino acid sequence (FQNEAQ) set forth in SEQ ID NO: 19.

[0143] In certain embodiments, for example, the anti-MAGE-A1 TCR encoded by the first nucleotide sequence 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.

[0144] In some embodiments, the non-CDR regions of the alpha and / or beta chains of an epitope-specific TCR, such as an anti-MAGE-A1 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, such that the alpha and / or beta chain is not naturally occurring. In some embodiments, the substitutions or mutations may improve the TCR described herein in various aspects, e.g., binding affinity, binding specificity, stability, viscosity, or any combination thereof.

[0145] In some embodiments, for example, an anti-MAGE-A1 TCR encoded by a 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 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, for example, an anti-MAGE-A1 TCR encoded by a 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 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, where the anti-MAGE-A1 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, for example, the anti-MAGE-A1 TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain present in the alpha chain amino acid sequence shown in SEQ ID NO:11.

[0146] In some embodiments, for example, an anti-MAGE-A1 TCR encoded by a 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 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, for example, an anti-MAGE-A1 TCR encoded by a 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 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, where the anti-MAGE-A1 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:20. In some embodiments, for example, the anti-MAGE-A1 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:12.

[0147] In some embodiments, for example, the anti-MAGE-A1 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, for example, the anti-MAGE-A1 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 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, for example, an anti-MAGE-A1 TCR encoded by a 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 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, where the anti-MAGE-A1 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, for example, an anti-MAGE-A1 TCR encoded by a first nucleotide sequence comprises an alpha chain constant region present in the alpha chain amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, for example, an anti-MAGE-A1 TCR encoded by a first nucleotide sequence further comprises an alpha constant region that is distinct 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 contains at least one, at least two, at least three, at least four, or at least five 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.

[0148] In some embodiments, for example, an anti-MAGE-A1 TCR encoded by a 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 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, for example, an anti-MAGE-A1 TCR encoded by a 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 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, where the anti-MAGE-A1 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:20. In some embodiments, for example, an anti-MAGE-A1 TCR encoded by a first nucleotide sequence comprises a beta chain constant region present in the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, for example, an anti-MAGE-A1 TCR encoded by a first nucleotide sequence further comprises a beta constant region that is distinct 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 amino acid sequence of the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 12.

[0149] In certain embodiments, for example, an anti-MAGE-A1 TCR encoded by a 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 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, for example, an anti-MAGE-A1 TCR encoded by a 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 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 the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, for example, the anti-MAGE-A1 TCR encoded by the first nucleotide sequence comprises an alpha chain comprising the amino acid sequence shown in SEQ ID NO:11.

[0150] In certain embodiments, for example, an anti-MAGE-A1 TCR encoded by a 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, for example, an anti-MAGE-A1 TCR encoded by a 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, where the anti-MAGE-A1 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, for example, the anti-MAGE-A1 TCR encoded by the first nucleotide sequence comprises a beta chain comprising the amino acid sequence shown in SEQ ID NO:12.

[0151] In some embodiments, for example, an anti-MAGE-A1 TCR encoded by a first nucleotide sequence comprises an alpha chain constant region, a beta chain constant region, or both, where the alpha chain constant region, the beta chain constant region, or both, comprises an amino acid sequence that has at least one, at least two, at least three, at least four, or at least five substitutions in the target sequence relative to the corresponding amino acid sequence of an endogenous TCR. In some embodiments, for example, an anti-MAGE-A1 TCR encoded by a first nucleotide sequence comprises an alpha chain and a beta chain, where the alpha chain comprises a constant region and the beta chain comprises a constant region, where (i) the alpha chain constant region comprises an amino acid sequence that has at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the alpha chain that comprises the amino acid sequence set forth in SEQ ID NO:11, and (ii) the beta chain constant region comprises an amino acid sequence that has at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the beta chain that comprises the amino acid sequence set forth in SEQ ID NO:12.

[0152] In some embodiments, the anti-MAGE-A1 TCR cross-competes with the reference TCR for binding to human MAGE-A1. In some embodiments, the anti-MAGE-A1 TCR binds to the same epitope of human MAGE-A1 as the reference TCR or an overlapping epitope. In some embodiments, the reference TCR comprises an alpha chain and a beta chain, and the alpha chain of the reference TCR comprises the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the beta chain of the reference TCR comprises the amino acid sequence as set forth in SEQ ID NO: 12.

[0153] II.A.1.c. Anti-MART1 TCR In some embodiments, the epitope-specific TCR, e.g., the epitope-specific TCR encoded by the first nucleotide sequence, specifically binds to an epitope on human MART1 (an "anti-MART1 TCR"), and the anti-MART1 TCR comprises an alpha chain CDR3 that comprises the amino acid sequence (CAVYGGATNKLIF) set forth in SEQ ID NO: 27. In some embodiments, the anti-MART1 TCR comprises a beta chain CDR3 that comprises the amino acid sequence (CASSPHAGGVDEKLFF) set forth in SEQ ID NO: 30.

[0154] In some embodiments, for example, the anti-MART1 TCR encoded by the first nucleotide sequence comprises an alpha chain CDR1, which alpha chain CDR1 of the anti-MART1 TCR comprises the amino acid sequence (TRDTTYYL) set forth in SEQ ID NO: 25. In some embodiments, for example, the anti-MART1 TCR encoded by the first nucleotide sequence comprises a beta chain CDR1, which beta chain CDR1 of the anti-MART1 TCR comprises the amino acid sequence (KGHSH) set forth in SEQ ID NO: 28.

[0155] In some embodiments, for example, the anti-MART1 TCR encoded by the first nucleotide sequence comprises an alpha chain CDR2, which alpha chain CDR2 of the anti-MART1 TCR comprises the amino acid sequence (NVLDGL) set forth in SEQ ID NO: 26. In some embodiments, for example, the anti-MART1 TCR encoded by the first nucleotide sequence comprises a beta chain CDR2, which beta chain CDR2 of the anti-MART1 TCR comprises the amino acid sequence (LQKENI) set forth in SEQ ID NO: 29.

[0156] In a particular embodiment, for example, the anti-MART1 TCR encoded by the first nucleotide sequence 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.

[0157] In some embodiments, the non-CDR regions of the alpha and / or beta chains of an epitope-specific TCR, such as an anti-MART1 TCR, are further modified, e.g., 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, or 6 amino acids are substituted or mutated, such that the alpha and / or beta chain is not naturally occurring. In some embodiments, the substitutions or mutations may improve the TCRs described herein in various aspects, e.g., binding affinity, binding specificity, stability, viscosity, or any combination thereof.

[0158] In some embodiments, for example, 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 with the variable domain of the alpha chain amino acid sequence shown in SEQ ID NO: 21. In some embodiments, for example, 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 98%, or at least about 99% sequence identity with the variable domain of the alpha chain amino acid sequence shown in SEQ ID NO: 21, where the anti-MART1 TCR comprises an alpha chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, for example, the anti-MART1 TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain present in the alpha chain amino acid sequence shown in SEQ ID NO:21.

[0159] In some embodiments, for example, the anti-MART1 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 with the variable domain of the beta chain amino acid sequence shown in SEQ ID NO: 22. In some embodiments, for example, the anti-MART1 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 98%, or at least about 99% sequence identity with the variable domain of the beta chain amino acid sequence shown in SEQ ID NO: 22, where the anti-MART1 TCR comprises a beta chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, for example, the anti-MART1 TCR encoded by the first nucleotide sequence comprises a beta chain variable domain present in the amino acid sequence shown in SEQ ID NO:22.

[0160] In some embodiments, for example, the anti-MART1 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, for example, the anti-MART1 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 98%, at least about 99%, or about 100% sequence identity with the constant region of the alpha chain amino acid sequence shown in SEQ ID NO:21. In some embodiments, for example, an anti-MART1 TCR encoded by a 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 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 the amino acid sequence set forth in SEQ ID NO:27. The TCR comprises an alpha chain constant region present in the alpha chain amino acid sequence shown in SEQ ID NO: 21. In some embodiments, for example, the anti-MART1 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 one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the constant region of the alpha chain amino acid sequence shown in SEQ ID NO: 21.

[0161] In some embodiments, for example, the anti-MART1 TCR 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 with the constant region of the beta chain amino acid sequence shown in SEQ ID NO: 22. In some embodiments, for example, the anti-MART1 TCR 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 98%, or at least about 99% sequence identity with the constant region of the beta chain amino acid sequence shown in SEQ ID NO: 22, where the anti-MART1 TCR comprises a beta chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, for example, the anti-MART1 TCR encoded by the first nucleotide sequence comprises a beta chain constant region present in the amino acid sequence shown in SEQ ID NO: 22. In some embodiments, for example, the anti-MART1 TCR 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 amino acid sequence of the constant region of the beta chain amino acid sequence shown in SEQ ID NO: 22.

[0162] In certain embodiments, for example, the anti-MART1 TCR 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 with the alpha chain amino acid sequence shown in SEQ ID NO: 21. In some embodiments, for example, the anti-MART1 TCR 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 with the alpha chain amino acid sequence shown in SEQ ID NO: 21, where the anti-MART1 TCR comprises an alpha chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, for example, the anti-MART1 TCR encoded by the first nucleotide sequence comprises an alpha chain comprising the amino acid sequence shown in SEQ ID NO: 21.

[0163] In certain embodiments, for example, the anti-MART1 TCR 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 with the beta chain amino acid sequence shown in SEQ ID NO: 22. In some embodiments, for example, the anti-MART1 TCR 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 with the beta chain amino acid sequence shown in SEQ ID NO: 22, where the anti-MART1 TCR comprises a beta chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, for example, the anti-MART1 TCR encoded by the first nucleotide sequence comprises a beta chain comprising the amino acid sequence shown in SEQ ID NO: 22.

[0164] In some embodiments, for example, the anti-MART1 TCR encoded by the first nucleotide sequence comprises an alpha chain constant region, a beta chain constant region, or both, where the alpha chain constant region, the beta chain constant region, or both, comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five substitutions in the target sequence relative to the corresponding amino acid sequence of the endogenous TCR. The TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region and the beta chain comprises a constant region, wherein (i) the alpha chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the alpha chain 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 one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the beta chain comprising the amino acid sequence set forth in SEQ ID NO:22.

[0165] In some embodiments, for example, the anti-MART1 TCR encoded by the first nucleotide sequence cross-competes with the reference TCR for binding to human MART1. In some embodiments, the anti-MART1 TCR binds to the same epitope of human MART1 as the reference TCR or an overlapping epitope. In some embodiments, the reference TCR comprises an alpha chain and a beta chain, and the alpha chain of the reference TCR comprises the amino acid sequence as set forth in SEQ ID NO:21. In some embodiments, the beta chain of the reference TCR comprises the amino acid sequence as set forth in SEQ ID NO:22.

[0166] II.A.1.d. Anti-MAGE-A3 TCR In some embodiments, the epitope-specific TCR, for example the epitope-specific TCR encoded by the first nucleotide sequence, specifically binds to an epitope on human MAGE-A3 (an "anti-MAGE-A3 TCR"), and the anti-MAGE-A3 TCR comprises an alpha chain CDR3 that comprises the amino acid sequence (CALEVRSSASKIIF) set forth in SEQ ID NO: 37. In some embodiments, the anti-MAGE-A3 TCR comprises a beta chain CDR3 that comprises the amino acid sequence (CSANPRTTLYEQYF) set forth in SEQ ID NO: 40.

[0167] In some embodiments, for example, the anti-MAGE-A3 TCR encoded by the first nucleotide sequence comprises an alpha chain CDR1, which comprises the amino acid sequence (TRDTTYY) set forth in SEQ ID NO: 35. In some embodiments, for example, the anti-MAGE-A3 TCR encoded by the first nucleotide sequence comprises a beta chain CDR1, which comprises the amino acid sequence (DFQATT) set forth in SEQ ID NO: 38.

[0168] In some embodiments, for example, the anti-MAGE-A3 TCR encoded by the first nucleotide sequence comprises an alpha chain CDR2, which comprises the amino acid sequence (RNSFDEQN) set forth in SEQ ID NO: 36. In some embodiments, for example, the anti-MAGE-A3 TCR encoded by the first nucleotide sequence comprises a beta chain CDR2, which comprises the amino acid sequence (SNEGSKA) set forth in SEQ ID NO: 39.

[0169] In certain embodiments, for example, the anti-MAGE-A3 TCR encoded by the first nucleotide sequence 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.

[0170] In some embodiments, the non-CDR regions of the alpha and / or beta chains 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, such that the alpha and / or beta chain is not naturally occurring. In some embodiments, the substitutions or mutations may improve the TCR described herein in various aspects, e.g., binding affinity, binding specificity, stability, viscosity, or any combination thereof.

[0171] In some embodiments, for example, an anti-MAGE-A3 TCR encoded by a 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 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, for example, an anti-MAGE-A3 TCR encoded by a 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 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 the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, for example, the anti-MAGE-A3 TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain present in the alpha chain amino acid sequence shown in SEQ ID NO:31.

[0172] In some embodiments, for example, an anti-MAGE-A3 TCR encoded by a 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 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, for example, an anti-MAGE-A3 TCR encoded by a 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 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, where the anti-MAGE-A3 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, for example, the anti-MAGE-A3 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:32.

[0173] In some embodiments, for example, the anti-MAGE-A3 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, for example, the anti-MAGE-A3 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 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, for example, an anti-MAGE-A3 TCR encoded by a 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 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, where the anti-MAGE-A3 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, for example, an anti-MAGE-A3 TCR encoded by a first nucleotide sequence comprises an alpha chain constant region present in the alpha chain amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, for example, an anti-MAGE-A3 TCR encoded by a first nucleotide sequence further comprises an alpha constant region that is distinct 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 contains at least one, at least two, at least three, at least four, or at least five 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.

[0174] In some embodiments, for example, an anti-MAGE-A3 TCR encoded by a 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 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, for example, an anti-MAGE-A3 TCR encoded by a 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 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, where the anti-MAGE-A3 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, for example, an anti-MAGE-A3 TCR encoded by a first nucleotide sequence comprises a beta chain constant region present in the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, for example, an anti-MAGE-A3 TCR encoded by a first nucleotide sequence further comprises a beta constant region that is distinct 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 one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 32.

[0175] In certain embodiments, for example, an anti-MAGE-A3 TCR encoded by a 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 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, for example, an anti-MAGE-A3 TCR encoded by a 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 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO: 31, where the anti-MAGE-A3 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, for example, the anti-MAGE-A3 TCR encoded by the first nucleotide sequence comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO:31.

[0176] In certain embodiments, for example, an anti-MAGE-A3 TCR encoded by a 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, for example, an anti-MAGE-A3 TCR encoded by a 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 the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, for example, the anti-MAGE-A3 TCR encoded by the first nucleotide sequence comprises a beta chain comprising the amino acid sequence set forth in SEQ ID NO:32.

[0177] In some embodiments, for example, the anti-MAGE-A3 TCR 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, comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five substitutions within the target sequence relative to the corresponding amino acid sequence of the endogenous TCR.

[0178] In some embodiments, for example, the anti-MAGE-A3 TCR encoded by the first nucleotide sequence comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region and the beta chain comprises a constant region, wherein (i) the alpha chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the alpha chain 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 one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the beta chain comprising the amino acid sequence set forth in SEQ ID NO:32.

[0179] In some embodiments, for example, the anti-MAGE-A3 TCR encoded by the first nucleotide sequence cross-competes with the reference TCR for binding to human MAGE-A3. 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 the amino acid sequence set forth in SEQ ID NO:31. In some embodiments, the beta chain of the reference TCR comprises the amino acid sequence as set forth in SEQ ID NO:32.

[0180] II.A.1.e. Anti-SSX2 TCR In some embodiments, an epitope-specific TCR, e.g., an epitope-specific TCR encoded by a first nucleotide sequence, specifically binds to an epitope on human SSX2 (an "anti-SSX2 TCR"), and the anti-SSX2 TCR comprises an alpha chain CDR3 that comprises the amino acid sequence (CAVEPMEYGNKLVF) set forth in SEQ ID NO: 47. In some embodiments, the anti-SSX2 TCR comprises a beta chain CDR3 that comprises the amino acid sequence (CASSALFSGANVLTF) set forth in SEQ ID NO: 50.

[0181] In some embodiments, for example, the anti-SSX2 TCR encoded by the first nucleotide sequence comprises an alpha chain CDR1, the alpha chain CDR1 of the anti-SSX2 TCR comprises the amino acid sequence (DSAIYN) set forth in SEQ ID NO: 45. In some embodiments, for example, the anti-SSX2 TCR encoded by the first nucleotide sequence comprises a beta chain CDR1, the beta chain CDR1 of the anti-SSX2 TCR comprises the amino acid sequence (LNHDA) set forth in SEQ ID NO: 48.

[0182] In some embodiments, for example, the anti-SSX2 TCR encoded by the first nucleotide sequence comprises an alpha chain CDR2, which comprises the amino acid sequence (IQSSQRE) set forth in SEQ ID NO: 46. In some embodiments, for example, the anti-SSX2 TCR encoded by the first nucleotide sequence comprises a beta chain CDR2, which comprises the amino acid sequence (SQIVND) set forth in SEQ ID NO: 49.

[0183] In certain embodiments, for example, the anti-SSX2 TCR 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.

[0184] In some embodiments, the non-CDR regions of the alpha and / or beta chains of an epitope-specific TCR, such as an anti-SSX2 TCR, are further modified, e.g., by substitution or mutation of one, two, three, four, five, or six amino acids, such that the alpha and / or beta chain is not naturally occurring. In some embodiments, the substitution or mutation may improve the TCR described herein in various aspects, e.g., binding affinity, binding specificity, stability, viscosity, or any combination thereof.

[0185] In some embodiments, for example, an anti-SSX2 TCR encoded by a 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, for example, an anti-SSX2 TCR encoded by a 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 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, where the anti-SSX2 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, for example, the anti-SSX2 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:41.

[0186] In some embodiments, for example, an anti-SSX2 TCR encoded by a 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, for example, an anti-SSX2 TCR encoded by a 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 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, where the anti-SSX2 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50. In some embodiments, for example, the anti-SSX2 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:42.

[0187] In some embodiments, for example, the anti-SSX2 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, for example, the anti-SSX2 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 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, for example, an anti-SSX2 TCR encoded by a 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 98%, or at least about 99% sequence identity with the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO:41, where the anti-SSX2 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, for example, an anti-SSX2 TCR encoded by a 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, for example, an anti-SSX2 TCR encoded by a first nucleotide sequence further comprises an alpha constant region that is distinct 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 one, at least two, at least three, at least four, or at least five 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.

[0188] In some embodiments, for example, the anti-SSX2 TCR 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, for example, the anti-SSX2 TCR 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 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, where the anti-SSX2 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50. In some embodiments, for example, the anti-SSX2 TCR encoded by the first nucleotide sequence comprises a beta chain constant region present in the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, for example, the anti-SSX2 TCR 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 one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 42.

[0189] In certain embodiments, for example, the anti-SSX2 TCR 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, for example, the anti-SSX2 TCR 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, where the anti-SSX2 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, for example, the anti-SSX2 TCR encoded by the first nucleotide sequence comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 41.

[0190] In certain embodiments, for example, the anti-SSX2 TCR 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, for example, the anti-SSX2 TCR 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, where the anti-SSX2 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50. In some embodiments, for example, the anti-SSX2 TCR encoded by the first nucleotide sequence comprises a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 42.

[0191] In some embodiments, for example, the anti-SSX2 TCR encoded by the first nucleotide sequence comprises an alpha chain constant region, a beta chain constant region, or both, where the alpha chain constant region, the beta chain constant region, or both, comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five substitutions in the target sequence relative to the corresponding amino acid sequence of the endogenous TCR. In some embodiments, for example, the anti-SSX2 TCR encoded by the first nucleotide sequence comprises an alpha chain and a beta chain, where the alpha chain comprises a constant region, and the beta chain comprises a constant region, where (i) the alpha chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the alpha chain 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 one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the beta chain comprising the amino acid sequence set forth in SEQ ID NO:42.

[0192] In some embodiments, for example, the anti-SSX2 TCR encoded by the first nucleotide sequence cross-competes with the reference TCR for binding to human SSX2. In some embodiments, the anti-SSX2 TCR binds to the same epitope of human SSX2 as the reference TCR or an overlapping epitope. In some embodiments, the reference TCR comprises an alpha chain and a beta chain, and the alpha chain of the reference TCR comprises the amino acid sequence set forth in SEQ ID NO:41. In some embodiments, the beta chain of the reference TCR comprises the amino acid sequence as set forth in SEQ ID NO:42.

[0193] II.A.2 Second Nucleotide Sequence The second nucleotide sequence of the nucleic acid molecule disclosed herein may be any sequence or may encode any polypeptide capable of inhibiting 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 the nucleotide sequence encoding the constant region of an endogenous TCR. In certain embodiments, the one or more siRNAs are complementary to a target sequence within the nucleotide sequence encoding the constant region of a wild-type, human TCR. In some embodiments, the one or more siRNAs are complementary to a target sequence within the nucleotide sequence encoding the 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 the nucleotide sequence encoding the constant region of a beta chain of a wild-type TCR. In some embodiments, the one or more siRNAs include (i) one or more siRNAs complementary to a target sequence within the nucleotide sequence encoding the constant region of an alpha chain of a wild-type TCR, and (ii) one or more siRNAs complementary to a target sequence within the nucleotide sequence encoding the constant region of a beta chain of a wild-type TCR.

[0194] 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, a 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 the constant region of the alpha chain of a wild-type TCR, wherein the one or more siRNAs comprise the nucleic acid sequence set forth in SEQ ID NOs: 57 and 58. [Table 4]

[0195] 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 target sequences within the nucleotide sequence encoding the constant region of the beta chain of the wild type TCR, 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 comprising the nucleic acid sequences set forth in SEQ ID NOs: 57 and 58, which are complementary to target sequences within the nucleotide sequence encoding the constant region of the alpha chain of the wild type TCR, and (ii) one or more siRNAs comprising the nucleic acid sequences set forth in SEQ ID NOs: 59 and 60, which are complementary to target sequences within the nucleotide sequence encoding the constant region of the beta chain of the wild type TCR.

[0196] In some embodiments, the second nucleotide sequence of the nucleic acid molecule comprises SEQ ID NO: 57-60. In some embodiments, the second nucleotide sequence comprises SEQ ID NO: 57-60, where one or more of SEQ ID NO: 57-60 are separated by one or more nucleic acids that do not encode an siRNA. In certain embodiments, the one or more siRNAs are selected from the siRNAs disclosed in U.S. Publication No. 2010 / 0273213A1, which is incorporated herein by reference in its entirety.

[0197] In some embodiments, the second nucleotide sequence of the nucleic acid molecule encodes a protein, where the protein can inhibit expression of an endogenous, e.g., wild-type, TCR. In some embodiments, the second nucleotide sequence encodes Cas9.

[0198] II.A.3 Vectors 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 a virus. In some embodiments, the vector is a mammalian vector. In some embodiments, the vector is a bacterial vector.

[0199] 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 vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, and an adeno-associated virus (AAV) vector. In certain embodiments, the vector is an AAV vector. In some embodiments, the vector is a lentivirus. In certain 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 may be used in the present disclosure can be found in Huang and Kamihira, Biotechnol.Adv.31(2):208-23(2103), which is incorporated herein by reference in its entirety.

[0200] II.B. Recombinant T Cell Receptors (TCRs) 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 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.

[0201] The epitope-specific TCR can be selected from (i) any epitope-specific TCR disclosed herein, e.g., any TCR disclosed in Sections IIA.1.a to II.A.1.e above, and (ii) any TCR that cross-competes for binding to a target human protein with a reference antibody selected from any epitope-specific TCR disclosed herein, e.g., any TCR disclosed in Sections IIA.1.a to II.A.1.e above.

[0202] 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.

[0203] 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.

[0204] In a particular embodiment, the epitope-specific TCR is an anti-MART1 TCR as disclosed herein. In a particular embodiment, 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.

[0205] 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.

[0206] 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.

[0207] II.B.3. Bispecific T cell receptor (TCR) Certain aspects of the disclosure relate to bispecific TCRs comprising a first antigen binding domain and a second antigen binding domain, the first antigen binding domain comprising 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, the first antigen binding domain comprising 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, the first antigen binding domain comprising 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, the first antigen binding domain comprising 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, the first antigen binding domain comprising 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, the first antigen-binding domain comprising an anti-SSX2 TCR or an antigen-binding portion thereof disclosed herein. In some embodiments, the first antigen-binding domain comprises a single chain variable fragment ("scFv").

[0208] 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 not 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 CD3, CD2, CD5, CD6, CD8, CD11a (LFA-1α), 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 an scFv.

[0209] In some embodiments, the first and second antigen-binding domains are linked or attached by a covalent bond, hi some embodiments, the first and second antigen-binding domains are linked by a peptide bond.

[0210] II.C. Epitope 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 human tyrosinase (an "anti-tyrosinase TCR"), and the anti-tyrosinase TCR binds to an epitope of tyrosinase that comprises the amino acid sequence set forth in SEQ ID NO:51 (FQDYIKSYL). In some embodiments, the anti-tyrosinase TCR binds to an epitope of tyrosinase consisting of the amino acid sequence set forth in SEQ ID NO:51. In some embodiments, the epitope is amino acid residues 460-468 of tyrosinase (SEQ ID NO:89), e.g., "tyrosinase 460―468In some embodiments, the epitope comprises amino acid residues 460-468 of tyrosinase (SEQ ID NO:89), e.g., "tyrosinase 460―468 " It consists of.

[0211] In some embodiments, the antigen-specific TCR specifically binds human MAGE-A1 (an "anti-MAGE-A1 TCR"), and the anti-MAGE-A1 TCR binds to an epitope of MAGE-A1 that comprises 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 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 comprises amino acid residues 289-297 of MAGE-A1 (SEQ ID NO: 90), e.g., "MAGE-A1 289-297 " It consists of.

[0212] In some embodiments, the antigen-specific TCR specifically binds human MART1 ("anti-MART1 TCR"), and 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 set forth in SEQ ID NO: 53. In some embodiments, the epitope consists of amino acid residues 25-33 of MART1 (SEQ ID NO: 91), e.g., "MART1 25-33 In some embodiments, the epitope comprises amino acid residues 25-33 of MART1 (SEQ ID NO: 91), e.g., "MART1 25-33 " It consists of.

[0213] In some embodiments, the antigen-specific TCR specifically binds human MAGE-A3 (an "anti-MAGE-A3 TCR"), and the anti-MAGE-A3 TCR binds to an epitope of MAGE-A3 that includes 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 set forth in SEQ ID NO:54. In some embodiments, the epitope is amino acid residues 167-176 of MAGE-A3 (SEQ ID NO:92), e.g., "MAGE-A3 167-176 In some embodiments, the epitope comprises amino acid residues 167-176 of MAGE-A3 (SEQ ID NO: 92), e.g., "MAGE-A3 167-176 " It consists of.

[0214] In some embodiments, the antigen-specific TCR specifically binds to human SSX2 (an "anti-SSX2 TCR"), where the anti-SSX2 TCR binds to an epitope of SSX2 that comprises 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 set forth in SEQ ID NO:55. In some embodiments, the epitope is amino acid residues 41-49 of SSX2 (SEQ ID NO:93), e.g., "SSX2 41-49 In some embodiments, the epitope comprises amino acid residues 41-49 of SSX2 (SEQ ID NO:93), e.g., "SSX2 41-49 " It consists of.

[0215] II.D. HLA class I molecules Certain aspects of the present disclosure relate to complexes comprising the HLA class I molecules and epitopes disclosed herein. The HLA class I molecules can be any HLA class I molecules known in the art. In some embodiments, the HLA class I molecules are selected from HLA-A, HLA-B, and HLA-C alleles. In some embodiments, the HLA class I molecules are selected from HLA-E, HLA-F, and HLA-G alleles. In certain embodiments, the HLA class I molecules are HLA-A alleles. In certain embodiments, the HLA class I molecules are HLA-B alleles. In certain embodiments, the HLA class I molecules are HLA-C alleles.

[0216] Many HLA-A, HLA-B, and HLA-C alleles are known in the art, and any known allele may be used in the present disclosure. An updated list of HLA alleles is available at hla.alleles.org / (last visited February 27, 2019).

[0217] II.D.1. HLA-A alleles and their complexes Certain aspects of the present disclosure relate to complexes comprising an HLA class I molecule and an epitope, where the HLA class I molecule is an HLA-A allele and 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.

[0218] 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.

[0219] 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.

[0220] The default settings for HLA-A are HLA-A*02:01:01:01, HLA-A*02:01:01:02L, HLA -A*02:01:01:03、SONG-A*02:01:01:04、SONG-A*02:01:01:05、SONG-A*02:0 :01:06、SONG-A*02:01:01:07、SONG-A*02:01:01:08、SONG-A*02:01:01:09、H LA-A*02:01:01:10, SONG-A*02:01:01:11, SONG-A*02:01:01:12, SONG-A*02: 01:01:13、SONG-A*02:01:01:14、SONG-A*02:01:01:15、SONG-A*02:01:01:1 、SONG-A*02:01:01:17、SONG-A*02:01:01:18、SONG-A*02:01:01:19、SONG-A*0 2:01:01:20、SONG-A*02:01:01:21、SONG-A*02:01:01:22、SONG-A*02:01:01: 23、SONGS-A*02:01:01:24、SONGS-A*02:01:01:25、SONGS-A*02:01:01:26、SONGS-A* 02:01:01:27、SONGS-A*02:01:01:28、SONGS-A*02:01:01:29、SONGS-A*02:01:01 :30、SONGS-A*02:01:01:31、SONGS-A*02:01:01:32、SONGS-A*02:01:01:33、SONGS- A*02:01:01:34、SONG-A*02:01:01:35、SONG-A*02:01:01:36、SONG-A*02:01: 01:37、SONG-A*02:01:01:38、SONG-A*02:01:01:39、SONG-A*02:01:01:40、SONG -A*02:01:01:41、SONG-A*02:01:01:42、SONG-A*02:01:01:43、SONG-A*02:0 :01:44、SONG-A*02:01:01:45、SONG-A*02:01:01:46、SONG-A*02:01:01:47、H LA-A*02:01:01:48, SONG-A*02:01:01:49, SONG-A*02:01:01:50, SONG-A*02: 01:01:51、SONG-A*02:01:01:52、SONG-A*02:01:01:53、SONG-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、H LA-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*02:01:48 :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,H LA-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、HL A-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、HLA-A*02:103、HLA-A*02:104、HLA-A*02:105、HLA-A*02:106、HLA -A*02:107、HLA-A*02:108、HLA-A*02:109、HLA-A*02:110、HLA-A*02:111、HLA-A*02:112、HLA-A*02:113:01N、HLA-A*02:113:02N、HLA-A*02:114、HLA-A*02:115、HLA-A*02:116、HLA-A*02:117、HLA-A*02:118、HLA-A*02:119、HLA-A*02:11:01:01、HLA-A*02:11:01:02、HLA-A*02:11:02、HLA-A*02:11: 03、HLA-A*02:11:04、HLA-A*02:11:05、HLA-A*02:11:06、HLA-A*02:11:07、HLA-A*02:11:08、HLA-A*02:11:09、HLA-A*02:12、HLA-A*02:120、HLA-A*02:121、HLA-A*02:122、HLA-A*02:123、HLA-A*02:124、HLA-A*02:125N、HLA-A*02:126、HLA-A*02:127、HLA-A*02:128、HLA-A*02:129、HLA-A*02:13、HL A-A*02:130、HLA-A*02:131:01、HLA-A*02:131:02、HLA-A*02:132、HLA-A*02:133、HLA-A*02:134、HLA-A*02:135、HLA-A*02:136、HLA-A*02:137、HLA-A*02:138、HLA-A*02:139、HLA-A*02:14、HLA-A*02:140、HLA-A*02:141、HLA-A*02:142、HLA-A*02:143、HLA-A*02:144、HLA-A*02:145、HLA-A*02:146、HLA-A*02:147、HLA-A*02:148、HLA-A*02:149、HLA-A*02:150:01、HLA-A*02:150:02、HLA-A*02:151、HLA-A*02:152、HLA-A*02:153:01、HLA-A*02:153:02、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*02:15 9、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、H LA-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*02:1 92、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、HL A-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、HLA-A*02:266、HLA-A*02:267、HLA-A*02:268、HLA-A*02:269、HLA-A *02:27、HLA-A*02:270、HLA-A*02:271、HLA-A*02:272、HLA-A*02:273、HLA-A*02:274、HLA-A*02:275、HLA-A*02:276、HLA-A*02:277、HLA-A*02:278、HLA-A*02:279、HLA-A*02:2 8、HLA-A*02:280、HLA-A*02:281、HLA-A*02:282、HLA-A*02:283、HLA-A*02:284N、HLA-A*02:285、HLA-A*02:286、HLA-A*02:287、HLA-A*02:288、HLA-A*02:289:01、HLA-A*02:28 9:02、HLA-A*02:29、HLA-A*02:290、HLA-A*02:291、HLA-A*02:292、HLA-A*02:293Q、HLA-A*02:294、HLA-A*02:295、HLA-A*02:296、HLA-A*02:297、HLA-A*02:298、HLA-A*02:299、 HLA-A*02:300, HLA-A*02:301N, HLA-A*02:302, HLA-A*02:303, HLA-A*02:304, HLA-A*02:305N, HLA-A*02:306, HLA-A*02:307, HLA-A*02:308, HLA-A*02:309, HLA-A*02:30:01, HLA-A*02:30:02, HLA-A*02:31, HLA-A*02:310, HLA-A*02:311, HLA-A*02:312, HLA-A*02:313, HLA-A*02:314N, HLA-A*02:3 15、HLA-A*02:316、HLA-A*02:317、HLA-A*02:318、HLA-A*02:319、HLA-A*02:320、HLA-A*02:321N、HLA-A*02:322、HLA-A*02:323、HLA-A*02:324、HLA-A*02:325、HLA-A*02:326、HLA-A*02:327、HLA-A*02:328、HLA-A*02:329、HLA-A*02:32N、HLA-A*02:33、HLA-A*02:330、HLA-A*02:331、HLA-A*02:332、 HLA-A*02:333、HLA-A*02:334、HLA-A*02:335、HLA-A*02:336、HLA-A*02:337、HLA-A*02:338、HLA-A*02:339、HLA-A*02:34、HLA-A*02:340、HLA-A*02:341、HLA-A*02:342、HLA-A*02:343、HLA-A*02:344、HLA-A*02:345、HLA-A*02:346、HLA-A*02:347、HLA-A*02:348、HLA-A*02:349、HLA-A*02:350N、HLA -A*02:351、HLA-A*02:352、HLA-A*02:353、HLA-A*02:354、HLA-A*02:355、HLA-A*02:356N、HLA-A*02:357、HLA-A*02:358、HLA-A*02:359、HLA-A*02:35:01、HLA-A*02:35:02、HLA-A*02:35:03、HLA-A*02:36、HLA-A*02:360、HLA-A*02:361、HLA-A*02:362、HLA-A*02:363、HLA-A*02:364、HLA-A*02:365、HLA-A*02:366N, HLA-A*02:367, HLA-A*02:368, HLA-A*02:369, HLA-A*02:37, HLA-A*02:370, HLA-A*02:371, HLA-A*02:372, HLA-A*02:373N, HLA-A*02:374, HLA-A*02:375, HLA-A*02:376, HLA-A*02:377, HLA-A*02:378, HLA-A*02:379, HLA-A*02:38, HLA-A*02:380, HLA-A*02:381, HLA-A*02:382, HL A-A*02:383、HLA-A*02:384、HLA-A*02:385、HLA-A*02:386、HLA-A*02:387、HLA-A*02:388、HLA-A*02:389、HLA-A*02:39、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*0 2:45, HLA-A*02:450, HLA-A*02:451, HLA-A*02:452, HLA-A*02:453, HLA-A*02:454, HLA-A*02:455, HLA-A*02:456, HLA-A*02:457, HLA-A*02:458, HLA-A*02:459, HLA-A*02:46, HLA-A*02:460, HLA-A*02:461, HLA-A*02:462, HLA-A*02:463, HLA-A*02:464, HLA-A*02:465, HLA-A*02:466, HLA- A*02:467、HLA-A*02:468:01N、HLA-A*02:468:02N、HLA-A*02:469、HLA-A*02:47、HLA-A*02:470、HLA-A*02:471、HLA-A*02:472、HLA-A*02:473、HLA-A*02:474、HLA-A*02:475、HLA-A*02:476N、HLA-A*02:477、HLA-A*02:478、HLA-A*02:479、HLA-A*02:48、HLA-A*02:480、HLA-A*02:481、HLA-A *02:482、HLA-A*02:483、HLA-A*02:484、HLA-A*02:485、HLA-A*02:486、HLA-A*02:487、HLA-A*02:488、HLA-A*02:489、HLA-A*02:49、HLA-A*02:490N、HLA-A*02:491、HLA-A*02:492、HLA-A*02:493、HLA-A*02:494、HLA-A*02:495、HLA-A*02:496、HLA-A*02:497、HLA-A*02:498、HLA-A*02:499、 HLA-A*02:50、HLA-A*02:500Q、HLA-A*02:501N、HLA-A*02:502、HLA-A*02:503、HLA-A*02:504、HLA-A*02:505、HLA-A*02:506N、HLA-A*02:507、HLA-A*02:508、HLA-A*02:509、HLA-A*02:51、HLA-A*02:510、HLA-A*02:511、HLA-A*02:512、HLA-A*02:513、HLA-A*02:514N、HLA-A*02:515、HLA-A*02:51 6N、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*02:53 1. 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*02:54 8、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、HLA-A*02:576、HLA-A*02:577、HLA-A*02:578、HLA-A*02:579、HLA-A*02:58、HLA-A*02 :580、HLA-A*02:581、HLA-A*02:582、HLA-A*02:583、HLA-A*02:584、HLA-A*02:585、HLA-A*02:586、HLA-A*02:587、HLA-A*02:588、HLA-A*02:589、HLA-A*02:59、HLA-A*02:590、HLA-A*02:591:01、HLA-A*02:591:02、HLA-A*02:592、HLA-A*02:593、HLA-A*02:594、HLA-A*02:595、HLA-A*02:596、HLA-A* 02:597、HLA-A*02:598、HLA-A*02:599、HLA-A*02:600、HLA-A*02:601、HLA-A*02:602、HLA-A*02:603、HLA-A*02:604、HLA-A*02:605Q、HLA-A*02:606、HLA-A*02:607、HLA-A*02:608N、HLA-A*02:609、HLA-A*02:60:01、HLA-A*02:60:02、HLA-A*02:61、HLA-A*02:610:01、HLA-A*02:610:02、HLA-A*02:61 1、HLA-A*02:612、HLA-A*02:613、HLA-A*02:614、HLA-A*02:615、HLA-A*02:616、HLA-A*02:617、HLA-A*02:618Q、HLA-A*02:619、HLA-A*02:62、HLA-A*02:620、HLA-A*02:621、HLA-A*02:622N、HLA-A*02:623、HLA-A*02:624、HLA-A*02:625、HLA-A*02:626、HLA-A*02:627、HLA-A*02:628、HLA-A*02:629、HLA-A*02:63、HLA-A*02:630、HLA-A*02:631、HLA-A*02:632、HLA-A*02:633、HLA-A*02:634、HLA-A*02:635、HLA-A*02:636、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*02:78 3、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、HL A-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, HL A-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、The antibody is selected from the group consisting of 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.

[0221] II.D.2. HLA-B Alleles and Their Complexes Certain aspects of the present disclosure relate to a complex comprising an HLA class I molecule and an epitope, where the HLA class I molecule is an HLA-B allele and 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.

[0222] Another aspect of the present disclosure relates to a complex comprising an HLA class I molecule and an epitope, where the HLA class I molecule is an HLA-B allele and the epitope is a MART1 epitope disclosed herein. In a particular embodiment, the MART1 epitope comprises, consists of, or consists essentially of SEQ ID NO:53.

[0223] Another aspect of the disclosure relates to a complex comprising an HLA class I molecule and an epitope, wherein the HLA class I molecule is an HLA-B allele and the epitope is a MAGE-A3 epitope disclosed herein. In a particular embodiment, the MAGE-A3 epitope comprises, consists of, or consists essentially of SEQ ID NO:54.

[0224] In some embodiments, the HLA-B alleles are 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*60, HLA-B*61, HLA-B*62, HLA-B*63, HLA-B*64, HLA-B*65, HLA-B*66, HLA-B*67, HLA-B*68, HLA-B*69, HLA-B*70, HLA-B*71, HLA-B*72, HLA-B*73, HLA-B*74, HLA-B*75, HLA-B*76, HLA-B*77, HLA-B*78, HLA-B*79, HLA-B*80, HLA-B*81, HLA-B*82, HLA-B*83, HLA-B*84, HLA-B*85, HLA-B*86, HLA-B*87, HLA-B*89, HLA-B*90, HLA-B*91, HLA-B*92, HLA *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*5 7, selected from 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.

[0225] II.D.2.a. HLA-B*07 alleles and their complexes In some embodiments, the HLA-B allele is the HLA-B*07 allele. In certain embodiments, the complex comprises an HLA-B*07 allele as disclosed herein and a MAGE-A1 epitope, e.g., an epitope comprising, consisting of, or consisting essentially of SEQ ID NO:52. In certain embodiments, the HLA-B allele is the HLA-B*07:02 allele. In certain embodiments, the HLA-B allele is the HLA-B*07:03 allele. In certain embodiments, the HLA-B allele is the HLA-B*07:04 allele. In certain embodiments, the HLA-B allele is the HLA-B*07:05 allele. In certain embodiments, the HLA-B allele is the HLA-B*07:06 allele. In a particular embodiment, the complex comprises the HLA-B*07:02 allele and a MAGE-A1 epitope comprising, consisting of, or consisting essentially of SEQ ID NO:52.

[0226] The default settings for HLA-B are HLA-B*07:02:01:01, HLA-B*07:02:01:02, HLA-B*07:02:01:01; B*07:02:01:03, SONG-B*07:02:01:04, SONG-B*07:02:01:05, SONG-B*07:02: 01:06, SONG-B*07:02:01:07, SONG-B*07:02:01:08, SONG-B*07:02:01:09, SONG-B*07:02:01:09, HL A-B*07:02:01:10, SONG-B*07:02:01:11, SONG-B*07:02:01:12, SONG-B*07:0 2:01:13、SONG-B*07:02:01:14、SONG-B*07:02:02、SONG-B*07:02:03、SONG-B* 07:02:04、SONGS-B*07:02:05、SONGS-B*07:02:06、SONGS-B*07:02:07、SONGS-B*0 :02:08、SONG-B*07:02:09、SONG-B*07:02:10、SONG-B*07:02:11、SONG-B*07:0 2:12、SONG-B*07:02:13、SONG-B*07:02:14、SONG-B*07:02:15、SONG-B*07:02:1 6, SONG-B*07:02:17, SONG-B*07:02:18, SONG-B*07:02:19, SONG-B*07:02:20, SONG-B*07:02:21, SONG-B*07:02:22, SONG-B*07:02:23, SONG-B*07:02:24, SONG-B*07:02:24, SONG-B*07:02:2 A-B*07:02:25, SONG-B*07:02:26, ​​SONG-B*07:02:27, SONG-B*07:02:28, SONG- B*07:02:29, SONG-B*07:02:30, SONG-B*07:02:31, SONG-B*07:02:32, SONG-B* 07:02:33、SONGS-B*07:02:34、SONGS-B*07:02:35、SONGS-B*07:02:36、SONGS-B*0 :02:37、SONG-B*07:02:38、SONG-B*07:02:39、SONG-B*07:02:40、SONG-B*07:0 2:41、SONG-B*07:02:42、SONG-B*07:02:43、SONG-B*07:02:44、SONG-B*07:02: 45、SONG-B*07:02:46、SONG-B*07:02:47、SONG-B*07:02:48、SONG-B*07:02:49、SONG-B*07:02:50, SONG-B*07:02:51, SONG-B*07:02:52, SONG-B*07:02:53, SONG-B*07:02:53, SONG A-B*07:02:54、SONGS-B*07:02:55、SONGS-B*07:02:56、SONGS-B*07:02:57、SONGS-B *07:02:58、SONGS-B*07:02:59、SONGS-B*07:02:60、SONGS-B*07:02:61、SONGS-B*0 7:02:62、SONGS-B*07:02:63、SONGS-B*07:02:64、SONGS-B*07:02:65、SONGS-B*07:0 2:66, SONG-B*07:02:67, SONG-B*07:02:68, SONG-B*07:02:69, SONG-B*07:02: 70、SONGS-B*07:02:71、SONGS-B*07:02:72、SONGS-B*07:02:73、SONGS-B*07:03、SONGS -B*07:04:01, SONG-B*07:04:02, SONG-B*07:05:01:01, SONG-B*07:05:01:0 、SONGS-B*07:05:01:03、SONGS-B*07:05:01:04、SONGS-B*07:05:02、SONGS-B*07:0 :03、SONG-B*07:05:04、SONG-B*07:05:05、SONG-B*07:05:06、SONG-B*07:05:0 7、SONGS-B*07:05:08、SONGS-B*07:05:09、SONGS-B*07:06:01、SONGS-B*07:06:02、H LA-B*07:06:03、SONGS-B*07:07:01、SONGS-B*07:07:02、SONGS-B*07:08:01、SONGS -B*07:08:02, SONG-B*07:09:01, SONG-B*07:09:02, SONG-B*07:10, SONG-B*07: 100、SONGS-B*07:101、SONGS-B*07:102、SONGS-B*07:103、SONGS-B*07:104、SONGS-B* 07:105, SONG-B*07:106, SONG-B*07:107, SONG-B*07:108, SONG-B*07:109, SONG- B*07:11, SONG-B*07:110, SONG-B*07:111, SONG-B*07:112, SONG-B*07:113, HL A-B*07:114, SONG-B*07:115, SONG-B*07:116, SONG-B*07:117, SONG-B*07:118SONG-B*07:119, SONG-B*07:12, SONG-B*07:120, SONG-B*07:121, SONG-B*07:1 2、SONGS-B*07:123、SONGS-B*07:124、SONGS-B*07:125、SONGS-B*07:126、SONGS-B*07: 127、SONGS-B*07:128、SONGS-B*07:129、SONGS-B*07:13、SONGS-B*07:130、SONGS-B*0 7:131、SONGS-B*07:132、SONGS-B*07:133、SONGS-B*07:134、SONGS-B*07:135、SONGS-B *07:136:01、SONGS-B*07:136:02、SONGS-B*07:137、SONGS-B*07:138、SONGS-B*07: 139:01, SONGS-B*07:139:02, SONGS-B*07:14, SONGS-B*07:140, SONGS-B*07:141, HL A-B*07:142, SONG-B*07:143, SONG-B*07:144, SONG-B*07:145, SONG-B*07:1 、SONGS-B*07:147、SONGS-B*07:148、SONGS-B*07:149、SONGS-B*07:15、SONGS-B*07:1 0, SONG-B*07:151:01, SONG-B*07:151:02, SONG-B*07:152, SONG-B*07:153, HL A-B*07:154, SONG-B*07:155, SONG-B*07:156, SONG-B*07:157, SONG-B*07:158 SONG-B*07:159, SONG-B*07:16, SONG-B*07:160, SONG-B*07:161, SONG-B*07:1 2, SONG-B*07:163, SONG-B*07:164, SONG-B*07:165, SONG-B*07:166, SONG-B*07: 167、SONGS-B*07:168、SONGS-B*07:169、SONGS-B*07:17、SONGS-B*07:170、SONGS-B*0 7:171, SONG-B*07:172, SONG-B*07:173, SONG-B*07:174, SONG-B*07:175, SONG-B *07:176, SONGS-B*07:177, SONGS-B*07:178, SONGS-B*07:179, SONGS-B*07:180, HL A-B*07:181, SONG-B*07:182, SONG-B*07:183, SONG-B*07:184, SONG-B*07:1SONG-B*07:186, SONG-B*07:187, SONG-B*07:188, SONG-B*07:189, SONG- B*07:18:01、SONGS-B*07:18:02、SONGS-B*07:19、SONGS-B*07:190、SONGS-B *07:191、SONGS-B*07:192、SONGS-B*07:193、SONGS-B*07:194、SONGS-B*07: 195、SONGS-B*07:196、SONGS-B*07:197、SONGS-B*07:198、SONGS-B*07:199、 SONG-B*07:20, SONG-B*07:200, SONG-B*07:201, SONG-B*07:202, SONG-B*07:2 3、SONGS-B*07:204、SONGS-B*07:205、SONGS-B*07:206、SONGS-B*07:207、SONGS-B*0 :208、SONGS-B*07:209、SONGS-B*07:21、SONGS-B*07:210、SONGS-B*07:211、SONGS-B* 07:212、SONGS-B*07:213、SONGS-B*07:214、SONGS-B*07:215、SONGS-B*07:216、SONGS -B*07:217、SONGS-B*07:218、SONGS-B*07:219、SONGS-B*07:220、SONGS-B*07:221、 SONG-B*07:222, SONG-B*07:223, SONG-B*07:224, SONG-B*07:225, SONG-B*07:2 26. SONG-B*07:227, SONG-B*07:228:01, SONG-B*07:228:02, SONG-B*07:229, H LA-B*07:22:01、SONGS-B*07:22:02、SONGS-B*07:23、SONGS-B*07:230、SONGS-B*0 :231、SONGS-B*07:232、SONGS-B*07:233、SONGS-B*07:234、SONGS-B*07:235、SONGS-B *07:236、SONGS-B*07:237、SONGS-B*07:238、SONGS-B*07:239、SONGS-B*07:24、SONGS -B*07:240, SONG-B*07:241, SONG-B*07:242, SONG-B*07:243, SONG-B*07:244 SONG-B*07:245, SONG-B*07:246, SONG-B*07:247, SONG-B*07:248, SONG-B*07:2 49、SONGS-B*07:25、SONGS-B*07:250、SONGS-B*07:251、SONGS-B*07:252、SONGS-B*0 :253、SONGS-B*07:254、SONGS-B*07:255、SONGS-B*07:256、SONGS-B*07:257、SONGS-B *07:258、SONGS-B*07:259、SONGS-B*07:26、SONGS-B*07:260、SONGS-B*07:261、SONGS -B*07:262, SONG-B*07:263, SONG-B*07:264, SONG-B*07:265, SONG-B*07:266SONG-B*07:267, SONG-B*07:268, SONG-B*07:269, SONG-B*07:27, SONG-B*07:2 0, SONG-B*07:271, SONG-B*07:272, SONG-B*07:273, SONG-B*07:274, SONG-B*0 :275、SONGS-B*07:276:01、SONGS-B*07:276:02、SONGS-B*07:277、SONGS-B*07:2 、SONGS-B*07:279、SONGS-B*07:28、SONGS-B*07:280、SONGS-B*07:281、SONGS-B*07:2 82、SONGS-B*07:283、SONGS-B*07:284、SONGS-B*07:285、SONGS-B*07:286、SONGS-B*0 7:287、SONGS-B*07:288、SONGS-B*07:289、SONGS-B*07:29、SONGS-B*07:290、SONGS-B *07:291、SONGS-B*07:292、SONGS-B*07:293、SONGS-B*07:294、SONGS-B*07:295、HL A-B*07:296, SONG-B*07:297, SONG-B*07:298, SONG-B*07:299, SONG-B*07:30, H LA-B*07:300、SONGS-B*07:301、SONGS-B*07:302、SONGS-B*07:303:01、SONGS-B*0 :303:02、SONGS-B*07:304、SONGS-B*07:305、SONGS-B*07:306、SONGS-B*07:307、HL A-B*07:308, SONG-B*07:309, SONG-B*07:31, SONG-B*07:310, SONG-B*07:311 SONG-B*07:312, SONG-B*07:313, SONG-B*07:314, SONG-B*07:315, SONG-B*07:3 16. SONG-B*07:317, SONG-B*07:318, SONG-B*07:319, SONG-B*07:32, SONG-B*0 :320、SONGS-B*07:321、SONGS-B*07:322、SONGS-B*07:323、SONGS-B*07:324、SONGS-B *07:325、SONGS-B*07:326、SONGS-B*07:327、SONGS-B*07:328、SONGS-B*07:329、HL A-B*07:330、SONGS-B*07:331、SONGS-B*07:332、SONGS-B*07:333、SONGS-B*07:334、SONG-B*07:335, SONG-B*07:336, SONG-B*07:337, SONG-B*07:338, SONG-B*07: 339、SONGS-B*07:33:01、SONGS-B*07:33:02、SONGS-B*07:33:03、SONGS-B*07:34、H LA-B*07:340、SONGS-B*07:341、SONGS-B*07:342、SONGS-B*07:343、SONGS-B*07:3 4、SONGS-B*07:345、SONGS-B*07:346、SONGS-B*07:347、SONGS-B*07:348、SONGS-B*0 :349、SONGS-B*07:35、SONGS-B*07:350、SONGS-B*07:351、SONGS-B*07:352、SONGS-B* 07:353、SONGS-B*07:354、SONGS-B*07:355、SONGS-B*07:356、SONGS-B*07:357、SONGS -B*07:358, SONG-B*07:36, SONG-B*07:37:01, SONG-B*07:37:02, SONG-B*07:3 8, SONG-B*07:39, SONG-B*07:40, SONG-B*07:41, SONG-B*07:42, SONG-B*07:43 SONG-B*07:44, SONG-B*07:45, SONG-B*07:46, SONG-B*07:47, SONG-B*07:48, H LA-B*07:49, SONG-B*07:50, SONG-B*07:51, SONG-B*07:52, SONG-B*07:53, SONG -B*07:54, SONG-B*07:55, SONG-B*07:56:01, SONG-B*07:56:02, SONG-B*07:5 、SONGS-B*07:58、SONGS-B*07:59、SONGS-B*07:60、SONGS-B*07:61、SONGS-B*07:62、H LA-B*07:63, SONG-B*07:64, SONG-B*07:65, SONG-B*07:66, SONG-B*07:67, SONG -B*07:68:01, SONG-B*07:68:02, SONG-B*07:68:03, SONG-B*07:69, SONG-B*0 :70、SONGS-B*07:71、SONGS-B*07:72、SONGS-B*07:73、SONGS-B*07:74、SONGS-B*07:7 5:01:01, SONG-B*07:75:01:02, SONG-B*07:76, SONG-B*07:77, SONG-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, H 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.

[0227] II.D.2.b. HLA-B*18 alleles and their complexes In some embodiments, the HLA-B allele is an HLA-B*18 allele. In certain embodiments, the complex comprises an HLA-B*18 allele as disclosed herein and a MART1 epitope, such as 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.

[0228] In some embodiments, the HLA-B allele is an HLA-B*18 allele. In particular embodiments, the complex comprises an HLA-B*18 allele as disclosed herein and a MAGE-A3 epitope, e.g., an epitope comprising, consisting of, or consisting essentially of SEQ ID NO:54. In particular 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.

[0229] HLA-B was not expressed in patients with HLA-B*18:01:01:01 1:01:02、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*18:01:01:09、HLA-10B.01:01:01:09、HLA-101:01:01:01:07、HLA-B 8:01:01:11、HLA-B*18:01:01:12、HLA-B*18:01:01:13、HLA-B*18:01:01:15、HLA-16、、HL 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 :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 :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、 19、HLA-B*18:01:20、HLA-B*18:01:21、HLA-B*18:01:22、HLA-B*18:01:24、HLA-B*18:01:24、HLA-25*18:01、 6、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 The HLA-B*18:01:32 has been destroyed in the circulation.

[0230] The fragmentation patterns of HLA-B are HLA-B*18:02, HLA-B*18:03:01, and HLA-B*18:03:0 2、SONGS-B*18:04:01、SONGS-B*18:04:02、SONGS-B*18:05:01:01、SONGS-B*18:05:0 1:02、SONGS-B*18:06、SONGS-B*18:07:01、SONGS-B*18:07:02、SONGS-B*18:08、SONGS -B*18:09, SONG-B*18:10, SONG-B*18:100, SONG-B*18:101, SONG-B*18:102, SONG -B*18:103, SONG-B*18:104, SONG-B*18:105, SONG-B*18:106, SONG-B*18:107 SONG-B*18:108, SONG-B*18:109, SONG-B*18:11, SONG-B*18:110, SONG-B*18:1 、SONGS-B*18:112、SONGS-B*18:113、SONGS-B*18:114、SONGS-B*18:115、SONGS-B*18: 116、SON-B*18:117、SON-B*18:118、SON-B*18:119、SON-B*18:120、SON-B*1 8:121, SONG-B*18:122, SONG-B*18:123, SONG-B*18:124, SONG-B*18:125, SONG- B*18:126, SONG-B*18:127, SONG-B*18:128, SONG-B*18:129, SONG-B*18:12:01 SONG-B*18:12:02, SONG-B*18:13, SONG-B*18:130, SONG-B*18:131:01:01, SONG -B*18:131:01:02、SONGS-B*18:132、SONGS-B*18:133、SONGS-B*18:134、SONGS-B*1 :135、SONG-B*18:136、SONG-B*18:137、SONG-B*18:138N、SONG-B*18:139、SONG- B*18:14, SONG-B*18:140, SONG-B*18:141, SONG-B*18:142, SONG-B*18:143, SONG -B*18:144, SONG-B*18:145, SONG-B*18:146, SONG-B*18:147, SONG-B*18:148, H LA-B*18:149, SONG-B*18:15, SONG-B*18:150, SONG-B*18:151, SONG-B*18:152SONG-B*18:153, SONG-B*18:154N, SONG-B*18:155, SONG-B*18:156:01:01, SONG-B*18:156:01:01 A-B*18:156:01:02, SONG-B*18:157:01:01, SONG-B*18:157:01:02, SONG-B*1 8:158, SONG-B*18:159, SONG-B*18:160, SONG-B*18:161, SONG-B*18:17N, SONG- B*18:18:01:01, SONG-B*18:18:01:02, SONG-B*18:19, SONG-B*18:20, SONG-B* 18:21、SONGS-B*18:22、SONGS-B*18:23N、SONGS-B*18:24、SONGS-B*18:25、SONGS-B*1 8:26, SONG-B*18:27, SONG-B*18:28, SONG-B*18:29, SONG-B*18:30, SONG-B*18: 31. SONG-B*18:32, SONG-B*18:33, SONG-B*18:34, SONG-B*18:35, SONG-B*18:3 、SONGS-B*18:37:01、SONGS-B*18:37:02、SONGS-B*18:38、SONGS-B*18:39、SONGS-B*1 8:40、SONGS-B*18:41、SONGS-B*18:42、SONGS-B*18:43、SONGS-B*18:44:01、SONGS-B *18:44:02、SONGS-B*18:45、SONGS-B*18:46、SONGS-B*18:47、SONGS-B*18:48、SONGS- B*18:49, SONG-B*18:50, SONG-B*18:51, SONG-B*18:52, SONG-B*18:53, SONG-B* 18:54, SONG-B*18:55, SONG-B*18:56, SONG-B*18:57:01, SONG-B*18:57:02, HL A-B*18:58, SONG-B*18:59, SONG-B*18:60, SONG-B*18:61, SONG-B*18:62, SONG- B*18:63, SONG-B*18:64, SONG-B*18:65, SONG-B*18:66, SONG-B*18:67, SONG-B* 18:68, SONG-B*18:69, SONG-B*18:70, SONG-B*18:71, SONG-B*18:72:01, SONG-B *18:72:02、SONG-B*18:72:03、SONG-B*18:73、SONG-B*18:74N、SONG-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*1 8:88, 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.

[0231] II.D.3. HLA-C alleles and their complexes Certain aspects of the present disclosure relate to complexes comprising an HLA class I molecule and an epitope, where the HLA class I molecule is an HLA-C allele and 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.

[0232] In some embodiments, the HLA-C allele is selected from the HLA-C*05:01 allele, the HLA-C*05:03 allele, the HLA-C*05:04 allele, the HLA-C*05:05 allele, and the HLA-C*05:06 allele. In certain embodiments, the HLA-C allele is the HLA-C*05:01 allele. In certain embodiments, the HLA-C allele is the HLA-C*05:03 allele. In certain embodiments, the HLA-C allele is the HLA-C*05:04 allele. In certain embodiments, the HLA-C allele is the HLA-C*05:05 allele. In certain embodiments, the HLA-C allele is the HLA-C*05:06 allele.

[0233] In certain embodiments, the complex comprises an HLA-C*05 allele as disclosed herein and a tyrosinase epitope, 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.

[0234] HLA-C was also defined as HLA-C*05:01:01:01:01 HLA-C*05:01:01:02. 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、HL A-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、H LA-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*05:0 1:18、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、HL A-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*0 5:01:39、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*05:13、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, HL A-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*05:181、HLA-C*05:182 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*05:44:01:0*0-20、HLA-HLA 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*05:58:58-AHL、HL C*05:59、HLA-C*05:60、HLA-C*05:61、HLA-C*05:62、HLA-C*05:6 3、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, which is not expressed in HLA-C*05:99N.

[0235] II.E.TCR Information Technology Certain aspects of the present disclosure relate to cells 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 may be used in the present disclosure.

[0236] In certain embodiments, the cell expresses CD3. Expression of CD3 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).

[0237] In some embodiments, the T cells are isolated from a human subject. In some embodiments, the human subject is the same subject that will ultimately receive the T cell therapy. In other embodiments, the subject is a donor subject, which is not the same subject that will receive the T cell therapy.

[0238] In some embodiments, the cell is a cell that does not naturally express CD3, where the cell is modified to express CD3. In some embodiments, the cell comprises a transgene encoding CD3, where the transgene is expressed by the cell. In some embodiments, the cell comprises a transgene encoding a protein that activates the expression of endogenous CD3 by the cell. In some embodiments, the cell comprises a transgene encoding a protein or siRNA that inhibits an inhibitor of CD3 expression in the cell. In some embodiments, the transgene is integrated into the genome of the cell. In some embodiments, the transgene is not integrated into the genome of the cell.

[0239] In some embodiments, the cells that are modified to express CD3 are isolated from a human subject. In some embodiments, the human subject is the same subject that will ultimately receive cell therapy. In other embodiments, the subject is a donor subject, and the donor subject is not the same subject that will ultimately receive cell therapy.

[0240] II.F. Vaccines Certain aspects of the present disclosure disclose a cancer vaccine comprising a peptide comprising the amino acid sequence 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.

[0241] III. Methods of the Disclosure 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 population of T cells obtained from a human subject.

[0242] III.A. Cancer Treatment Methods 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 above.

[0243] In some embodiments, the cancer is melanoma, bone cancer, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, cutaneous or intraocular malignant melanoma, pancreatic cancer, skin cancer, head and neck cancer, 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), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the parathyroid gland, The cancer is selected from renal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of these cancers. In some embodiments, the cancer is melanoma.

[0244] In some embodiments, the cancer is recurrent. In some embodiments, the cancer is refractory. In some embodiments, the cancer is progressive. In some embodiments, the cancer is metastatic.

[0245] In some embodiments, the methods disclosed herein treat cancer in a subject. In some embodiments, the methods disclosed herein reduce the severity of one or more symptoms of cancer. In some embodiments, the methods disclosed herein reduce the size or number of tumors resulting from cancer. In some embodiments, the methods disclosed herein extend the overall survival of a subject compared to a subject not provided with a method disclosed herein. In some embodiments, the methods disclosed herein increase the progression-free survival of a subject compared to a subject not provided with a method disclosed herein. In some embodiments, the methods disclosed herein result in a partial response in a subject. In some embodiments, the methods disclosed herein result in a complete remission in a subject.

[0246] In some embodiments, the methods disclosed herein include treating cancer in a subject in need thereof, comprising administering to the subject a cell described herein, the cell comprising 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 that has been modified to express CD3.

[0247] In some embodiments, the cells, e.g., T cells, are obtained from the subject. In some embodiments, the cells, e.g., T cells, are obtained from a donor other than the subject.

[0248] In some embodiments, the subject is pretreated prior to administering the cells. Pretreatment may include any substance that promotes T cell function and / or survival. In some embodiments, the pretreatment includes administering to the subject a chemotherapy, a cytokine, a protein, a small molecule, or any combination thereof. In some embodiments, the pretreatment includes administering an interleukin. In some embodiments, the pretreatment includes administering IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof. In some embodiments, the pretreatment includes administering cyclophosphamide, fludarabine, or both. In some embodiments, the pretreatment includes administering vitamin C, an AKT inhibitor, ATRA (vesanoid, tretinoin), rapamycin, or any combination thereof.

[0249] III.B. Methods for Engineering Antigen-Target Cells Certain aspects of the disclosure relate to methods of engineering antigen-targeted 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 includes 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 that has been modified to express CD3 as described herein. In some embodiments, the cell, e.g., a T cell, is obtained from a subject in need of T cell therapy. In some embodiments, the cell is obtained from a donor other than the subject in need of T cell therapy. In some embodiments, the cell is a T cell or a natural killer cell.

[0250] III. Methods for Enriching Target Populations of CT Cells Certain aspects of the present disclosure relate to a method for enriching a target population of T cells obtained from a human subject. In some embodiments, the method comprises contacting T cells with an HLA class I molecule as disclosed herein. In some embodiments, the method comprises contacting T cells with an APC as disclosed herein. In some embodiments, after contacting, the enriched population of T cells comprises a greater number of T cells capable of binding to HLA class I molecules relative to the number of T cells capable of binding to HLA class I molecules before contacting.

[0251] In some embodiments, the method comprises contacting T cells in vitro with a peptide comprising 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 method comprises contacting T cells in vitro with a peptide consisting 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, after contacting, the enriched population of T cells comprises a greater number of T cells capable of binding to HLA class I molecules relative to the number of T cells capable of binding to HLA class I molecules prior to contacting.

[0252] Some aspects of the present disclosure relate to a method of selecting T cells capable of targeting tumor cells. In some embodiments, the method comprises contacting a population of in vitro isolated T cells with a peptide consisting 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 obtained from a human subject.

[0253] The T cells obtained from the human subject can be any T cell disclosed herein. In some embodiments, the T cells obtained from the human subject are tumor infiltrating lymphocytes (TILs).

[0254] In some embodiments, the method further comprises administering the enriched T cells to a human subject, in some embodiments, the subject is pretreated as described herein prior to receiving the T cells.

[0255] All of the various aspects, embodiments, and options described herein may be combined in any and all variations.

[0256] 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.

[0257] Having generally described the present disclosure, a further understanding can be obtained by reference to the examples provided herein, which are for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0258] Example 1 - Method Cell samples

[0259] Peripheral blood samples were obtained from healthy donors. Mononuclear cells were obtained by density gradient centrifugation (Ficoll-Paque PLUS; GE Healthcare). K562 is an erythroleukemia cell line with defective HLA expression. T2 is HLA-A*02:01. + T-cell leukemia / B-LCL hybrid cell lines. Jurkat 76 is a T-cell leukemia cell line that lacks expression of TCR and CD8. K562, T2, and Jurkat 76 cell lines were cultured in RPMI 1640 supplemented with 10% FBS and 50 μg / ml gentamicin (Invitrogen). TILs isolated from metastatic melanoma patients were expanded in vitro.

[0260] Tyrosinase: Me275 and MCF7 cell lines were grown in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin (Invitrogen). Melme-3M cell line was grown in IMDM supplemented with 20% FBS and 50 μg / ml gentamicin (Invitrogen).

[0261] MAGE-A1:Me275, SK-MEL-37, and SK-MEL-21 cell lines were grown in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin (Invitrogen).

[0262] MART1:A375 and SK-MEL-28 cell lines were grown in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin (Invitrogen). Melme-3M cell line was grown in IMDM supplemented with 20% FBS and 50 μg / ml gentamicin.

[0263] MAGE-A3:SK-MEL-28 and HEK293T cell lines were grown in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin (Invitrogen).

[0264] SSX2:SK-MEL-21, SK-MEL-37, and SK-MEL-28 cell lines were grown in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin (Invitrogen).

[0265] peptide

[0266] The synthetic peptides were dissolved in DMSO at 50 μg / ml.

[0267] Tyrosinase: The peptide used was tyrosinase restricted to C*05:01. 460-468 (FQDYIKSYL; SEQ ID NO:51) and HIV rev 67-75 (SAEPVPLQL; SEQ ID NO: 82) peptide. As a negative control, HIV rev 67-75 peptide was used.

[0268] MAGE-A1: The peptide used was MAGE-A1 restricted to B*07:02. 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ー137 (TPGPGVRYPL; SEQ ID NO: 85) peptide. 60-72 , EBV EBNA3A 379-387 , and HIV nef 128ー137 The peptide was used as a negative control.

[0269] MART1: The peptides used were MART1 and MART1 restricted to B*18:01. 25-33 (EEAAGIGIL; SEQ ID NO:53), MAGE-A3 167-176 (MEVDPIGHLY; SEQ ID NO:54), and HIV gag 161-170 (FRDYVDRFYK; SEQ ID NO: 86) was a 20-mer overlapping peptide to cover the entire protein. MAGE-A3 167-176 and HIV gag 161-170 The peptide was used as a negative control.

[0270] MAGE-A3: The peptide used was MAGE-A3 restricted to B*18:01. 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) peptide. MART1 25-33 and HIV gag 161-170 The peptide was used as a negative control.

[0271] SSX2: The peptide used was SSX2 restricted to A*02:01. 41-49 (KASEKIFYV; SEQ ID NO:55), NY-ESO-1157-165 (SLLMWITQV; SEQ ID NO: 87), and HTLV-1 tax 11-19 (LLFGYPVYV; SEQ ID NO: 88) peptide. 157-165 and HTLV-1 tax 11-19 The peptide was used as a negative control.

[0272] gene

[0273] Where applicable, each of the HLA C*05:01, B*07:02, and B*18:01 genes was fused to a truncated form of the human nerve growth factor receptor (ΔNGFR) via an internal ribosome entry site. 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 the published sequence. The full-length MART1 gene was cloned from Malme-3M cells via RT-PCR according to the published sequence. The full-length SSX2 gene was cloned from SK-MEL-37 cells via RT-PCR according to the published sequence. TCR genes were cloned by 5'-rapid amplification of cDNA ends (RACE) PCR 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 the pMX retroviral vector and transduced using the 293GPG cell-based retroviral system.

[0274] Transformants (transfectants)

[0275] Jurkat 76 / CD8 cells were transduced with individual TCRα and TCRβ genes. TCR transfectants derived from Jurkat 76 / CD8 were purified (purity >95%) using CD3 Microbeads (Miltenyi Biotec). K562-based artificial APCs expressing various HLA class I genes individually as single HLA alleles in combination with CD80 and CD83 have been reported previously (Butler and Hirano, Immunol. Rev. 257:191-209 (2014); Hirano et al., Clin. Cancer Res. 12:2967-75 (2006)). Retroviral supernatant from PG13 was used to transduce TCR genes into human primary T cells. TransIT293 (Mirus Bio) was used to transfect TCR genes into the 293GPG cell line.

[0276] Tyrosinase - MCF7 cells were retrovirally transduced with the full-length tyrosinase gene to generate MCF7 / tyrosinase cells. Expression of the transduced tyrosinase was assessed by flow cytometry after staining with anti-tyrosinase monoclonal antibody (mAb) (clone ERP10141; Abcam). HLA-C*05:01 - Malme-3M and Me275 cells were retrovirally transduced with HLA-C*05:01 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 + The cells were purified (>95% pure) and used in subsequent experiments. The ΔNGFR gene alone was retrovirally transduced as a control.

[0277] MAGE-A1 -SK-MEL-21 cells were retrovirally transduced with the full-length MAGE-A1 gene to generate SK-MEK-21 / MAGE-A1 cells. Expression of transduced MAGE-A1 was assessed by flow cytometry after staining with anti-MAGE-A1 mAb (clone MA454; LifeSpan Biosciences). HLA-B*07:02 - Me275 and SK-MEL-37 cells were retrovirally transduced with HLA-B*07:02 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 + The cells were purified (>95% pure) and used in subsequent experiments. The ΔNGFR gene alone was retrovirally transduced as a control.

[0278] MART1 - A375 cells were retrovirally transduced with the full-length MART1 gene to generate A375 / MART1 cells. Expression of transduced MART1 was assessed 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 were retrovirally transduced with HLA-B*18:01 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 the ΔNGFR + The cells were purified (>95% pure) and used in subsequent experiments. The ΔNGFR gene alone was retrovirally transduced as a control.

[0279] MAGE-A3 -HEK293T cells were retrovirally transduced with the full-length MAGE-A3 gene to generate HEK293T / MAGE-A3 cells. Expression of MAGE-A3 in the transduced cells was assessed by Western blot analysis using anti-MAGE-A3 polyclonal antibody (pAb) (LifeSpan Biosciences). HLA-B*18:01 - SK-MEL-28 and HEK293T cells were retrovirally transduced with HLA-B*18:01 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 + The cells were purified (>95% pure) and used in subsequent experiments. The ΔNGFR gene alone was retrovirally transduced as a control.

[0280] SSX2 - SK-MEL-21 and SK-MEL-28 cells were retrovirally transduced with the full-length SSX2 gene to generate SK-MEK-21 / SSX2 and SK-MEL-28 / SSX2 cells. Expression of SSX2 in transduced cells was assessed by Western blot analysis using anti-SSX2 pAb (Thermo Fisher Scientific). HLA-A*02:01 - SK-MEL-28 cells were retrovirally transduced with HLA-A*02:01 to generate SK-MEL-28 / A*02:01 cells.

[0281] Flow cytometry and cell sorting

[0282] Cell surface molecules were identified using PC5-conjugated anti-CD8 mAb (clone B9.11; Beckman Cells were stained with LIVE / DEAD Fixable Aqua Dead Cell Stain kit (Life Technologies), FITC-conjugated anti-NGFR (clone ME20.4; Biolegend), and APC / Cy7-conjugated anti-CD3 (clone UCHT1; Biolegend). Dead cells were identified with the LIVE / DEAD Fixable Aqua Dead Cell Stain 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 a FACS Aria II (BD Bioscience).

[0283] Cytokine ELISPOT analysis

[0284] IFN-γ ELISPOT assays were 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 capture mAb (1-D1K; MABTECH, Mariemont, OH) and T cells were cultured at 2 × 10 per well with or without peptide. 4The plates were incubated with 1000 target cells for 20–24 h at 37°C. Plates were then washed and incubated with biotin-conjugated detection mAb (7-B6-1; MABTECH). HRP-conjugated SA (Jackson ImmunoResearch) was then added to generate IFN-γ spots. The reaction was stopped by thorough rinsing 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).

[0285] CD8 in an HLA-restricted peptide-specific manner + Expansion of TILs

[0286] Where applicable, e.g., for MAGE-A3 TCR, CD8 + TILs are CD8 + TILs were purified by negative magnetic selection using a T Cell Isolation Kit (Miltenyi Biotec). B*18:01 artificial APCs were pulsed with 10 μg / mL of the class I-restricted peptide of interest for 6 h. Artificial APCs were then irradiated at 200 Gy, washed, and added to TILs at an effector to target (E:T) ratio of 20:1. Starting 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 3 days.

[0287] Primary CD8 transduced with cloned TCR + T cell proliferation

[0288] CD3 +T cells were purified by negative magnetic selection using a Pan T Cell Isolation Kit (Miltenyi Biotec). Purified T cells were stimulated with 200 Gy irradiated artificial APC / mOKT3 at an E:T ratio of 20:1. Starting the next day, activated T cells were retrovirally transduced with the cloned TCR genes by centrifugation at 1,000 g for 1 h 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. Culture medium was replenished every 2–3 days.

[0289] Mammalian cell-based production of pHLA multimers

[0290] The affinity matured HLA class I genes were modified to contain a Glu (E) residue instead of a Gln (Q) residue at position 115 in the α2 domain and a mouse K b By fusing the extracellular domain of the affinity-matured HLA class I gene with a Gly-Ser (GS) flexible linker followed by a 6xHis tag, we have produced a soluble HLA class I Q115E -K b HEK293T cells were transfected with various soluble HLA class I antigens along with the β2m gene using a 293GPG cell-based retroviral system. Q115E -K b The genes were individually transduced with soluble affinity maturation class I Q115E -K b Stable HEK293T cells ectopically expressing HLA class I were grown to confluence and then their medium was replaced. After 48 hours, conditioned medium was harvested and either used immediately or frozen until further use. Soluble HLA class I produced by HEK293T transfectants Q115E -K b The containing supernatant was incubated overnight at 37°C with 100-1000 μg / ml of the class I restricted peptide of interest for in vitro peptide exchange. Q115E -Kb dimerized using anti-His mAb (clone AD1.1.10; Abcam) conjugated to a fluorescent dye such as phycoerythrin (PE) at a molar ratio of 2:1 for 2 h at room temperature, or overnight at 4 °C. Functional soluble HLA class I Q115E -K b The concentration of the 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 Abs, respectively.

[0291] pHLA multimer staining

[0292] T cells (1×10 5 ) in the presence of 50 nM dasatinib (LC laboratories) for 30 min at 37 °C. Cells were then washed and incubated with 5–10 μg / ml multimer for 30 min at room temperature, and AffiniPure Fab fragment goat anti-mouse IgG1 conjugated to R-phycoerythrin (Jackson ImmunoResearch Laboratories) was added for 15 min at 4 °C. Cells were then washed three times and co-stained with anti-CD8 mAb for 15 min at 4 °C. Dead cells were finally identified using the LIVE / DEAD Fixable Aqua Dead Cell Stain kit.

[0293] statistical analysis

[0294] Statistical analysis was performed using GraphPad Prism 5.0e. Welch's t-test (two-tailed) analysis was performed to determine whether two groups were significantly different for a particular variable. P values ​​less than 0.05 were considered significant.

[0295] Example 2 - Tyrosinase-specific TCR Tumor infiltrating lymphocytes (TILs) were isolated from metastatic melanoma patients and then polyclonally expanded in vitro to examine their tyrosinase antigen specificity for the HLA-C*05:01 allele. A combination of structure-based and functional assays using peptide / HLA (pHLA) multimers has been used to measure antigen-specific T cell responses. T cells express tyrosinase 460-468 TILs were stained using pHLA multimers bearing the peptide C*05:01 / tyrosinase (Figure 1). 460-468 Multimer-positive T cells secreted detectable IFN-γ in an HLA-restricted peptide-specific manner by ELISPOT analysis (Figure 2).

[0296] Multimer-positive antitumor T cells were collected and their TCR genes were molecularly cloned (Figure 3). The antigen specificity and functional reactivity of the cloned TCR were verified by multimer staining and ELISPOT assay of TCR-reconstituted T cells. When reconstituted with primary T cells, C*05:01 / tyrosinase 460-468 TCR-transduced T cells were successfully stained with cognate multimers (Figure 4), and tyrosinase, as indicated by surface C*05:01 molecules. 460-468 Both melanoma cell lines reacted strongly with the peptide (Figure 5). Importantly, these cells were able to recognize C*05:01-matched and peptide-unpulsed tumor cells that naturally express the tyrosinase gene. Malme-3M and Me275 melanoma cell lines are negative for C*05:01, although they do endogenously express the tyrosinase gene. When the C*05:01 molecule was ectopically expressed, both melanoma cell lines showed no C*05:01 / tyrosinase activity. 460-468 C*05:01 / tyrosinase was successfully recognized by TCR-transduced T cells. Furthermore, MCF7 breast cancer cells, which lack endogenous expression of tyrosinase, were successfully recognized by C*05:01 / tyrosinase when transduced with the full-length tyrosinase gene. 460-468 These results suggest that C*05:01 / tyrosinase 460-468TCR-transduced T cells have sufficient affinity to recognize tumor cells, and the cloned C*05:01 / tyrosinase 460-468 This clearly demonstrates that TCR is tumor reactive.

[0297] Example 3 - MAGE-A1 specific TCR TILs were isolated from patients with metastatic melanoma and then polyclonally expanded in vitro to examine their MAGE-A1 antigen specificity for the HLA-B*07:02 allele. 289-297 TILs were stained using pHLA multimers bearing the peptide (Figure 9). 289-297 Multimer-positive T cells secreted detectable IFN-γ in an HLA-restricted peptide-specific manner by ELISPOT analysis (Figure 10).

[0298] Multimer-positive anti-tumor T cells were collected and their TCR genes were molecularly cloned (Figure 11). The antigen specificity and functional reactivity of the cloned TCR were verified by multimer staining and ELISPOT assay of TCR-reconstituted T cells. When reconstituted with primary T cells, B*07:02 / MAGE-A1 289-297 TCR-transduced T cells were successfully stained with cognate multimers (Figure 12), and MAGE-A1 was expressed by surface B*07:02 molecules. 289-297 Both Me275 and SK-MEL-37 melanoma cell lines were negative for B*07:02, although they do endogenously express the MAGE-A1 gene. When the B*07:02 molecule was ectopically expressed, both melanoma cell lines were negative for B*07:02 / MAGE-A1. 289-297 Furthermore, SK-MEL-21 melanoma cells, which lack endogenous expression of MAGE-A1, were successfully recognized by TCR-transduced T cells when transduced with the full-length MAGE-A1 gene. 289-297These results suggest that B*07:02 / MAGE-A1 may be a potential treatment for TCR-transduced T cells. 289-297 TCR-transduced T cells have sufficient affinity to recognize tumor cells, and the cloned B*07:02 / MAGE-A1 289-297 TCR has been clearly shown to be tumor reactive.

[0299] The use of the newly cloned tumor-reactive B*07:02-restricted MAGE-A1 TCR gene may broaden the scope of application of anti-MAGE-A1 TCR gene therapy.

[0300] Example 4 - MART1-specific TCR TILs were isolated from metastatic melanoma patients and then polyclonally expanded in vitro to examine their MART1 antigen specificity for the HLA-B*18:01 allele. Because pHLA multimer production requires the use of peptides with known precise sequences, it is not straightforward or practical to perform high-throughput screening of new epitope peptides using a pHLA multimer-based strategy. In addition to structure-based analysis using pHLA multimers, functional analysis may be applied to determine the antigen specificity of T cells. We performed functional assays using artificial antigen-presenting cells (APCs) as stimulator cells, which can take up and process longer peptides and present epitope peptides via class I molecules. HLA-B*18:01 artificial APCs were pulsed with overlapping peptides to cover the entire protein of MART1 (Table 5) and used as stimulators in cytokine ELISPOT assays. When stimulated with B*18:01 artificial APCs pulsed with overlapping peptides derived from MART1, B*18:01 + Melanoma TILs expressed the shared sequence 21 YTTAEEAAGIGILTV 35 A series of mutant deletion peptides were used to identify the minimal peptide epitope presented by the B*18:01 molecule, i.e., 25 EEAAGIGIL33 Importantly, B*18:01 / MART1 25-33 Multimers successfully stained up to 9.2% of polyclonally expanded TILs, which was consistent with B*18:01 / MART1 25-33 These results suggest that T cells were the major population of TILs (Figure 18). Multimer-positive T cells secreted detectable IFN-γ in an HLA-restricted peptide-specific manner by ELISPOT analysis (Figure 19). [Table 5]

[0301] Multimer-positive anti-tumor T cells were collected and their TCR genes were molecularly cloned (Figure 20). The antigen specificity and functional reactivity of the cloned TCR were verified by multimer staining and ELISPOT assay of TCR-reconstituted T cells. When reconstituted with primary T cells, B*18:01 / MART1 25-33 TCR-transduced T cells were successfully stained with cognate multimers (Figure 21), indicated by surface B*18:01 molecules, MART1 25-33 Both melanoma cell lines, Malme-3M and SK-MEL-28, were negative for B*18:01, although they do endogenously express the MART1 gene. When the B*18:01 molecule was ectopically expressed, both melanoma cell lines were negative for B*18:01 / MART1. 25-33 Furthermore, A375 melanoma cells, which lack endogenous expression of both B*18:01 and MART1, were successfully recognized by TCR-transduced T cells only when transduced with both B*18:01 and the full-length MART1 gene (but not either of the single genes). 25-33 These results suggest that B*18:01 / MART1 cells responded to TCR-transduced T cells (Figures 23-25). 25-33TCR-transduced T cells have sufficient affinity to recognize tumor cells, and the cloned B*18:01 / MART1 25-33 TCR has been clearly shown to be tumor reactive.

[0302] The use of the newly cloned tumor-reactive B*18:01-restricted MART1 TCR gene may broaden the scope of anti-MART1 TCR gene therapy beyond HLA-A *02:01-positive cancer patients.

[0303] Example 5 - MAGE-A3 specific TCR TILs were isolated from metastatic melanoma patients and then polyclonally expanded in vitro to examine their MAGE-A3 antigen specificity for the HLA-B*18:01 allele. 167-176 Staining was performed using pHLA multimers bearing the peptide B*18:01 / MAGE-A3 in polyclonally expanded TILs. 167-176 The percentage of TILs positive for MAGE-A3 was only 0.04% before peptide-specific stimulation. 167-176 When weakly stimulated once with peptide-pulsed B*18:01 artificial APCs, 5.5% of TILs stained with the cognate multimers, B*18:01 / MAGE-A3 167-176 They secreted IFN-γ in a specific manner (Figures 26-27).

[0304] 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 TCR were verified by multimer staining and ELISPOT assay of TCR-reconstituted T cells. When reconstituted with primary T cells, B*18:01 / MAGE-A3 167-176 TCR-transduced T cells were successfully stained with cognate multimers (Figure 29), which are expressed by surface B*18:01 molecules, MAGE-A3 167-176The cells reacted strongly with the peptide (Figure 30). Importantly, these cells were able to recognize B*18:01-matched and peptide-unpulsed tumor cells that naturally express the MAGE-A3 gene. SK-MEL-28 melanoma cells are negative for B*18:01, but they do endogenously express the MAGE-A3 gene. When the B*18:01 molecule was ectopically expressed, melanoma cells were able to recognize the B*18:01 / MAGE-A3 167-176 Furthermore, HEK293T melanoma cells, which lack endogenous expression of both B*18:01 and MAGE-A3, were successfully recognized by TCR-transduced T cells only when transduced with both B*18:01 and the full-length MAGE-A3 gene (but not either of the single genes). 167-176 These results suggest that B*18:01 / MAGE-A3 is a novel T cell-specific T cell that is highly responsive to TCR-transduced T cells (Figures 31-33). 167-176 TCR-transduced T cells have sufficient affinity to recognize tumor cells, and the cloned B*18:01 / MAGE-A3 167-176 TCR has been clearly shown to be tumor reactive.

[0305] The use of the newly cloned tumor-reactive B*18:01 / MAGE-A3 TCR gene may broaden the scope of application of anti-MAGE-A3 TCR gene therapy beyond HLA-A*02:01-positive cancer patients.

[0306] Example 6 - SSX2-specific TCR TILs were isolated from patients with metastatic melanoma and then polyclonally expanded in vitro to examine their SSX2 antigen specificity for the HLA-A*02:01 allele. 41-49 The TILs were stained using pHLA multimers bearing the peptide (Figure 34). 41-49 Multimer-positive T cells secreted detectable IFN-γ in an HLA-restricted peptide-specific manner by ELISPOT analysis (FIG. 35).

[0307] Multimer-positive anti-tumor T cells were collected and their TCR genes were molecularly cloned (Figure 36). The antigen specificity and functional reactivity of the cloned TCR were verified by multimer staining and ELISPOT assay of TCR-reconstituted T cells. When reconstituted with primary T cells, A*02:01 / SSX2 41-49 TCR-transduced T cells were successfully stained with cognate multimers (Figure 37), indicated by surface A*02:01 molecules, SSX2 41-49 The cells reacted strongly with the peptide (Figure 38). Importantly, these cells were able to recognize A*02:01-matched and peptide-unpulsed tumor cells that naturally express the SSX2 gene. Although SK-MEL-21 melanoma cells are positive for A*02:01, they do not endogenously express the SSX2 gene. When the SSX2 gene was ectopically expressed, melanoma cells reacted strongly with A*02:01 / SSX2 41-49 Furthermore, SK-MEL-28 melanoma cells, which lack endogenous expression of both A*02:01 and SSX2, were successfully recognized by TCR-transduced T cells only when transduced with both A*02:01 and the full-length SSX2 gene (but not either of the single genes). 41-49 These results suggest that A*02:01 / SSX2 41-49 TCR-transduced T cells have sufficient affinity to recognize tumor cells, and the cloned A*02:01 / SSX2 41-49 TCR has been clearly shown to be tumor reactive.

[0308] The use of the newly cloned tumor-reactive A*02:01-restricted SSX2 TCR gene may broaden the scope of application of anti-SSX2 TCR gene therapy. In certain embodiments, for example, the following items are provided: (Item 1) 1. 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, (a) the target human protein is tyrosinase (an "anti-tyrosinase TCR"), said anti-tyrosinase TCR cross-competing for binding to human tyrosinase with a reference anti-tyrosinase TCR comprising an alpha chain and a beta chain, said alpha chain comprising the amino acid sequence set forth in SEQ ID NO:1 and said beta chain comprising the amino acid sequence set forth in SEQ ID NO:2; (b) the target human protein is MAGE-A1 (an "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 comprising an alpha chain and a beta chain, wherein the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:11 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:12; (c) the target human protein is MART1 ("anti-MART1 TCR"), said anti-MART1 TCR cross-competing for binding to human MART1 with a reference anti-MART1 TCR comprising an alpha chain and a beta chain, said alpha chain comprising the amino acid sequence set forth in SEQ ID NO:21 and said beta chain comprising the amino acid sequence set forth in SEQ ID NO:22; (d) the target human protein is MAGE-A3 (an "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 comprising an alpha chain and a beta chain, wherein the alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 31 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO: 32; or (e) The nucleic acid molecule, wherein the target human protein is SSX2 ("anti-SSX2 TCR"), the anti-SSX2 TCR cross-competes for binding to human SSX2 with a reference anti-SSX2 TCR comprising an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 41, and the beta chain comprising the amino acid sequence set forth in SEQ ID NO: 42. 1. 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, (a) the target human protein is tyrosinase (an "anti-tyrosinase TCR"), wherein the anti-tyrosinase TCR binds to the same epitope as, or an overlapping epitope of, human tyrosinase as a reference anti-tyrosinase TCR comprising an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO:1 and the beta chain comprising the amino acid sequence set forth in SEQ ID NO:2; (b) the target human protein is MAGE-A1 (an "anti-MAGE-A1 TCR"), wherein the anti-MAGE-A1 TCR binds to the same epitope as, or an overlapping epitope on, human MAGE-A1 as, a reference anti-MAGE-A1 TCR comprising an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO:11 and the beta chain comprising the amino acid sequence 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 as, or an overlapping epitope of, human MART1 as a reference anti-MART1 TCR comprising an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO:21 and the beta chain comprising the amino acid sequence set forth in SEQ ID NO:22; (d) the target human protein is MAGE-A3 (an "anti-MAGE-A3 TCR"), wherein the anti-MAGE-A3 TCR binds to the same epitope as, or an overlapping epitope of, human MAGE-A3 as, a reference anti-MAGE-A3 TCR comprising an alpha chain and a beta chain, wherein the alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 31 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO: 32; or (e) the nucleic acid molecule wherein the target human protein is SSX2 ("anti-SSX2 TCR"), the anti-SSX2 TCR binds to the same epitope as or an overlapping epitope of human SSX2 as a reference anti-SSX2 TCR comprising an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 41 and the beta chain comprising the amino acid sequence set forth in SEQ ID NO: 42. (Item 3) (a) the anti-tyrosinase TCR binds to an epitope of tyrosinase consisting of the amino acid sequence shown in SEQ ID NO:51; (b) the anti-MAGE-A1 TCR binds to an epitope of MAGE-A1 consisting of the amino acid sequence shown in SEQ ID NO:52; (c) the anti-MART1 TCR binds to an epitope of MART1 consisting of the amino acid sequence shown in SEQ ID NO:53; (d) the anti-MAGE-A3 TCR binds to an epitope of MAGE-A3 consisting of the amino acid sequence set forth in SEQ ID NO: 54; or (e) The nucleic acid molecule according to item 1 or 2, wherein the anti-SSX2 TCR binds to an epitope of SSX2 consisting of the amino acid sequence set forth in SEQ ID NO: 55. (Item 4) 4. The nucleic acid molecule according to item 2 or 3, wherein the epitope is complexed with an HLA class I molecule. (Item 5) 5. The nucleic acid molecule of item 4, wherein the HLA class I molecule is an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G allele. (Item 6) (a) the target human protein is tyrosinase and the HLA class I molecule is the HLA-C*05 allele; (b) the target human protein is MAGE-A1 and the HLA class I molecule is the HLA-B*07 allele; (c) the target human protein is MART1 and the HLA class I molecule is the HLA-B*18 allele; (d) the target human protein is MAGE-A3 and the HLA class I molecule is the HLA-B*18 allele; or (e) The nucleic acid molecule according to item 4, wherein the target human protein is SSX2 and the HLA class I molecule is an HLA-A*02 allele. (Item 7) (a) the target human protein is tyrosinase and the HLA class I molecule is selected from the HLA-C*05:01 allele, the HLA-C*05:03 allele, the HLA-C*05:04 allele, the HLA-C*05:05 allele, and the HLA-C*05:06 allele; (b) the target human protein is MAGE-A1 and the HLA class I molecule is selected from the HLA-B*07:02 allele, the HLA-B*07:03 allele, the HLA-B*07:04 allele, the HLA-B*07:05 allele, and the HLA-B*07:06 allele; (c) the target human protein is MART1 and the HLA class I molecule is selected from the HLA-B*18:01 allele, the HLA-B*18:02 allele, the HLA-B*18:03 allele, the HLA-B*18:04 allele, and the HLA-B*18:05 allele; (d) the target human protein is MAGE-A3 and the HLA class I molecule is selected from the HLA-B*18:01 allele, the HLA-B*18:02 allele, the HLA-B*18:03 allele, the HLA-B*18:04 allele, and the HLA-B*18:05 allele; or (e) The nucleic acid molecule according to any one of items 4 to 6, wherein the target human protein is SSX2 and the HLA class I molecule is selected from the HLA-A*02:01 allele, the HLA-A*02:02 allele, the HLA-A*02:03 allele, the HLA-A*02:04 allele, and the HLA-A*02:05 allele. (Item 8) (a) the target human protein is tyrosinase and the HLA class I molecule is the HLA-C*05:01 allele; (b) the target human protein is MAGE-A1 and the HLA class I molecule is the HLA-B*07:02 allele; (c) the target human protein is MART1 and the HLA class I molecule is the HLA-B*18:01 allele; (d) the target human protein is MAGE-A3 and the HLA class I molecule is the HLA-B*18:01 allele; or (e) The nucleic acid molecule according to any one of items 4 to 7, wherein the target human protein is SSX2 and the HLA class I molecule is the HLA-A*02:01 allele. (Item 9) 9. The nucleic acid molecule according to any one of claims 1 to 8, wherein the recombinant TCR or antigen-binding portion thereof that specifically binds to the target human protein comprises an alpha chain and a beta chain, the alpha chain comprising a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3, and the beta chain comprising a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; (a) the target human protein is tyrosinase and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:7; (b) the target human protein is MAGE-A1 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 17; (c) the target human protein is MART1 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:27; (d) the target human protein is MAGE-A3 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 37; or (e) the nucleic acid molecule, wherein the target human protein is SSX2 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:47. (Item 10) (a) the target human protein is tyrosinase and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10; (b) the target human protein is MAGE-A1 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20; (c) the target human protein is MART1 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30; (d) the target human protein is MAGE-A3 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40; or (e) The nucleic acid molecule of item 9, wherein the target human protein is SSX2 and the alpha beta CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50. (Item 11) 9. The nucleic acid molecule according to any one of claims 1 to 8, wherein the recombinant TCR or antigen-binding portion thereof that specifically binds to the target human protein comprises an alpha chain and a beta chain, the alpha chain comprising a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3, and the beta chain comprising a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; (a) the target human protein is tyrosinase and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10; (b) the target human protein is MAGE-A1 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20; (c) the target human protein is MART1 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30; (d) the target human protein is MAGE-A3 and the beta chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40; or (e) the nucleic acid molecule, wherein the target human protein is SSX2 and the alpha beta CDR3 comprises the amino acid sequence set forth in SEQ ID NO:50. (Item 12) (a) the target human protein is tyrosinase and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:7; (b) the target human protein is MAGE-A1 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 17; (c) the target human protein is MART1 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:27; (d) the target human protein is MAGE-A3 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 37; or (e) The nucleic acid molecule of claim 11, wherein the target human protein is SSX2 and the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 47. (Item 13) (a) the target human protein is tyrosinase and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:5; (b) the target human protein is MAGE-A1 and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15; (c) the target human protein is MART1 and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:25; (d) the target human protein is MAGE-A3 and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 35; or (e) The nucleic acid molecule of any one of items 9 to 12, wherein the target human protein is SSX2 and the alpha chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45. (Item 14) (a) the target human protein is tyrosinase and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8; (b) the target human protein is MAGE-A1 and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 18; (c) the target human protein is MART1 and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:28; (d) the target human protein is MAGE-A3 and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 38; or (e) The nucleic acid molecule of any one of items 9 to 13, wherein the target human protein is SSX2 and the beta chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 48. (Item 15) (a) the target human protein is tyrosinase and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:6; (b) the target human protein is MAGE-A1 and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 16; (c) the target human protein is MART1 and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:26; (d) the target human protein is MAGE-A3 and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 36; or (e) The nucleic acid molecule of any one of items 9 to 14, wherein the target human protein is SSX2 and the alpha chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 46. (Item 16) (a) the target human protein is tyrosinase and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9; (b) the target human protein is MAGE-A1 and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19; (c) the target human protein is MART1 and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:29; (d) the target human protein is MAGE-A3 and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 39; or (e) The nucleic acid molecule of any one of items 9 to 15, wherein the target human protein is SSX2 and the beta chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49. (Item 17) (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 the 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 the 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 the variable domain present in the amino acid sequence set forth in SEQ ID NO: 31; or (e) The nucleic acid molecule of any one of items 9 to 16, wherein the target human protein is SSX2 and the alpha chain variable domain comprises the amino acid sequence of the variable domain present in the amino acid sequence set forth in SEQ ID NO: 41. (Item 18) (a) the target human protein is tyrosinase and the beta chain variable domain comprises the amino acid sequence of the 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 the 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 the 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 the variable domain present in the amino acid sequence set forth in SEQ ID NO: 32; or (e) The nucleic acid molecule of any one of items 9 to 17, wherein the target human protein is SSX2 and the beta chain variable domain comprises the amino acid sequence of the variable domain present in the amino acid sequence set forth in SEQ ID NO: 42. (Item 19) 19. The nucleic acid molecule of any one of items 9 to 18, wherein the alpha chain further comprises a constant region, the constant region being distinct from an endogenous constant region of the alpha chain. (Item 20) 20. The nucleic acid molecule according to any one of items 9 to 19, wherein the alpha chain further comprises a constant region, (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 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 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 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 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 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 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 the constant region present in the amino acid sequence set forth in SEQ ID NO:31; or (e) the nucleic acid molecule, wherein 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 the constant region present in the amino acid sequence set forth in SEQ ID NO:41. (Item 21) (a) the target human protein is tyrosinase and the alpha chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five 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 having at least one, at least two, at least three, at least four, or at least five 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 having at least one, at least two, at least three, at least four, or at least five 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 having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO: 31; or (e) The nucleic acid molecule of item 19 or 20, wherein the target human protein is SSX2 and the alpha chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO: 41. (Item 22) 22. The nucleic acid molecule of any one of items 9 to 21, wherein the beta chain further comprises a constant region, the constant region being distinct from an endogenous constant region of the beta chain. (Item 23) 23. The nucleic acid molecule according to any one of items 9 to 22, wherein the beta chain further comprises a constant region, (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 the 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 the 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 the 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 the constant region present in the amino acid sequence set forth in SEQ ID NO: 32; or (e) the nucleic acid molecule, wherein 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 the constant region present in the amino acid sequence set forth in SEQ ID NO:42. (Item 24) (a) the target human protein is tyrosinase, and the beta chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the 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 one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the 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 one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the 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 one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO: 32; or (e) The nucleic acid molecule of any one of claims 22 to 23, wherein the target human protein is SSX2 and the beta chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO: 42. (Item 25) (a) the target human protein is tyrosinase and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:1; (b) the target human protein is MAGE-A1 and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:11; (c) the target human protein is MART1, and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 21; (d) the target human protein is MAGE-A3 and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 31; or (e) The nucleic acid molecule of any one of items 9 to 24, wherein the target human protein is SSX2 and the alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 41. (Item 26) (a) the target human protein is tyrosinase and the beta chain comprises the amino acid sequence shown in SEQ ID NO:2; (b) the target human protein is MAGE-A1 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO: 12; (c) the target human protein is MART1 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO: 22; (d) the target human protein is MAGE-A3 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO: 32; or (e) The nucleic acid molecule of any one of items 9 to 25, wherein the target human protein is SSX2 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO: 42. (Item 27) 27. The nucleic acid molecule of any one of items 1 to 26, wherein the second nucleotide sequence is one or more siRNAs that reduce expression of an endogenous TCR. (Item 28) 28. The nucleic acid molecule of claim 27, wherein the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of the endogenous TCR. (Item 29) 29. The nucleic acid molecule according to item 27 or 28, wherein the one or more siRNAs comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 57 to 60. (Item 30) 30. The nucleic acid molecule of any one of items 1 to 29, wherein the second nucleotide sequence encodes Cas9. (Item 31) 31. The nucleic acid molecule of any one of items 1 to 30, wherein the recombinant TCR or antigen-binding portion thereof comprises an alpha chain constant region, a beta chain constant region, or both, and the alpha chain constant region, the beta chain constant region, or both, comprises an amino acid sequence having a...

Claims

[Claim 1] The invention described in the specification.

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