MAGE-a4 t cell receptors and methods of use thereof

Abstract

To provide a MAGE-A4 T cell receptor and a method for using the same.SOLUTION: Provided are an isolated T cell receptor (TCR) that specifically binds to an HLA-presenting cancer testis antigen melanoma-related target antigen A4 (MAGE-A4) peptide, and a method of treatment and diagnosis using the isolated TCR. The T cell receptor (TCR) is a membrane-binding heterodimer including α chain and β chain which resemble an immunoglobulin variable (V) region and a constant (C) region.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 862,726, filed June 18, 2019, and U.S. Provisional Patent Application No. 62 / 871,793, filed July 9, 2019. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference herein in its entirety. The ASCII copy, created on June 15, 2020, is named 118003_00320_SL.txt and is 344,832 bytes in size. [Background technology]

[0003] T cell receptors (TCRs) are membrane-bound heterodimers containing α and β chains similar to immunoglobulin variable (V) and constant (C) regions. The TCR α chain contains a V-α chain covalently linked to a C-α chain, while the β chain contains a V-β chain covalently linked to a C-β chain. The V-α and V-β chains form a pocket or cleft that can bind antigen in the context of the major histocompatibility complex (MHC), known in humans as the HLA complex. (Davis Ann. Rev. of Immunology 3:537 (1985); Fundamental Immunology 3rd Ed., W. Paul Ed. New York (1993)).

[0004] The TCR is a key effector of the immune system, offering unique advantages as a platform for developing therapeutic agents. While antibody therapeutics are limited to recognizing pathogens in the blood and extracellular space or protein targets on the cell surface, T cell receptors can recognize antigens presented by MHC molecules on the cell surface, including antigens derived from intracellular proteins. Depending on the T cell subtype that recognizes and activates the presented antigen, TCRs can participate in regulating various immune responses. For example, T cells are involved in regulating humoral immune responses by inducing B cell differentiation into antibody-producing cells. Furthermore, activated T cells act to initiate cell-mediated immune responses. In addition, TCRs have been reported to mediate cell killing and increase B cell proliferation, affecting the development and severity of various disorders, including cancer, allergies, viral infections, and autoimmune disorders.

[0005] In terms of TCR function, antigen-specific TCRs are being evaluated for use in immunotherapy for their ability to redirect T cells to tumors expressing the antigen. TCRs bind small peptides, only 8–12 amino acids in length, which are bound on the surface of target cells by major histocompatibility complexes (MHC). Thus, TCRs can recognize intracellular antigens or viral proteins derived from cancer because these antigens are processed and presented as peptides in the context of surface MHC. Therefore, TCRs can recognize additional internal cellular targets that are not available to cell-penetrating antibodies or therapies.

[0006] However, a challenge in the industry is to engineer TCRs that lack immunogenicity when administered to patients and have fine specificity for a particular peptide antigen of interest without cross-reacting with similar epitopes found in other peptides on the MHC or in the natural protein repertoire.

[0007] MAGE-A4, or melanoma-associated antigen A4, is a well-known cancer-testis antigen (CTA) on the X chromosome. The function of MAGE-A4 is unknown, but it may be involved in cell cycle progression / regulation, transcriptional regulation, cell survival, and / or apoptosis. For example, overexpression of MAGE-A4 has been shown to promote proliferation of spontaneously transformed oral keratinocytes and inhibit cell growth arrest in G1 (Bhan, et al. (2012) Oncol Rep 28(4):1496).

[0008] MAGE-A4 is highly expressed by many tumors of different histological types, such as head and neck squamous cell carcinoma, lung cancers including non-small cell lung cancer, esophageal squamous cell carcinoma, colon cancer, bladder cancer, mucosal and cutaneous melanoma, ovarian cancers, e.g., serous carcinoma, and uterine cancer, but in normal healthy adult tissues, MAGE-A4 expression is restricted to the testis.

[0009] The ability of the MAGE-A4 antigen to induce immune responses together with its restricted expression pattern makes it an excellent candidate for cancer immunotherapy. There is an unmet need in the art for novel T cell receptor-based targeting agents that specifically bind to the MAGE-A4 antigen, and methods for producing and using such agents in therapeutic and diagnostic settings. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Davis Ann.Rev.of Immunology 3:537(1985) [Non-patent document 2] Fundamental Immunology 3rd Ed.,W.Paul Ed.New York(1993) [Non-patent document 3] Bhan, et al. (2012) Oncol Rep 28(4):1496 Summary of the Invention [Means for solving the problem]

[0011] The present invention provides T cell receptors (TCRs) directed against the MAGE-A4 peptide antigen in the context of MHC (HLA-A2). The unique TCR sequences identified show specific binding to the small peptide MAGE-A4, which resides in the groove of the HLA molecule, and demonstrate T cell activation in reporter assays. Furthermore, the TCRs of the present invention do not cross-react with other "similar" peptides.

[0012]

[0013] Accordingly, in one aspect, the present invention provides a T cell receptor (TCR) (e.g., an isolated TCR, or a TCR expressed on an isolated cell) that specifically binds to an HLA-A2-presented cancer-testis antigen melanoma associated antigen 4 (MAGE-A4) peptide comprising the amino acid sequence of KVLEHVVRV (SEQ ID NO: 609) (MAGE-A4 286-294), wherein the TCR comprises an alpha chain variable domain comprising complementarity-determining region (CDR) 3, and CDR3 has the structure of Formula I: N1-N2-N3-N4-N5-N6-N7-N8-N9-N 10 -N 11 -N 12 -N 13 -N 14 -N 15 (Formula I) comprising the amino acid sequence wherein N1 is a nonpolar amino acid; N2 is Val, but may be present or absent; N3 is Tyr, Gly, Leu, Val, Glu, Met, Ala, or Phe; N4 is Arg, Glu, Ser, Asn, Gln, Lys, Asp, Gly, or Met, but may be present or absent; N5 is Ser, Arg, Glu, Leu, Ala, Asp, Pro, Met, Gly, or Lys, but may be present or absent; N6 is Ala, Asp, Gly, Ser, Val, Pro, Leu, Tyr, or Thr, but may be present or absent; N7 is Thr, Pro, Ser, Glu, Asp, Trp, Arg, Asn, Ile, Gln, or Leu; N8 is His, Trp, Thr, Lys, Tyr, or Ala; N9 is Asn, Gly, Lys, Ile, Ser, or Arg; N 10 is Gln, Lys, Gly, Thr, Leu, Asp, or Ser, but may be present or absent; N 11 is Phe, Asn, Thr, Tyr, Ala, Leu, Met, or Glu, but may be present or absent; N 12 is Lys, Phe, Tyr, or Asp, but may be present or absent; N 13 is Lys or Gly, but may be present or absent, N 14 is Thr, Leu, or Tyr, but may be present or absent; N 15 is Tyr, Gln, Ile, Thr, Val, or Arg.

[0013] In one embodiment, N1 is Ala, Ile, or Gly.

[0014] In another aspect, the present invention provides a T cell receptor (TCR) that specifically binds to an HLA-A2-presented cancer-testis antigen melanoma associated antigen 4 (MAGE-A4) peptide comprising the amino acid sequence of KVLEHVVRV (SEQ ID NO: 609) (MAGE-A4 286-294), wherein the TCR comprises a beta chain variable domain comprising a complementarity determining region (CDR) 3, wherein CDR3 is represented by Formula II: N1-N2-N3-N4-N5-N6-N7-N8-N9-N 10 -N 11 -N 12 -N13 -N 14 -N 15 -N 16 -N 17 -N 18 (Formula II) comprising the amino acid sequence wherein N1 is Ala or Ser; N2 is Ala, Ser, or Thr; N3 is Ser, Gly, or Trp; N4 is Leu, Tyr, Trp, Asp, Phe, Gly, Pro, or His; N5 is Gly or Asp, but may be present or absent; N6 is Phe or Arg, but may be present or absent; N7 is Trp, Phe, Asp, Pro, Tyr, Gly, Thr, Ser, or Val, but may be present or absent; N8 is Pro, Arg, Asp, Tyr, Gln, Asn, or Gly, but may be present or absent; N9 is Asp but may be present or absent; N 10 is an Arg, which may or may not be present, N 11 is Gly, Ala, or Thr, but may be present or absent; N 12 is Ser, Trp, Thr, Gly, Val, Leu, Arg, Met, Tyr, or Gln, N 13 is Gly, but may be present or absent, N 14 is Asn, Asp, Gly, Thr, Pro, Gln, or His, but may be present or absent; N 15 is Thr, Ser, Glu, Asn, Tyr, Gln, Asp, or Pro, but may be present or absent, and N 16is Glu, Pro, Lys, Thr, Ala, Gly, or Gln, but may be present or absent; N 17 is Ala, Leu, Ile, Tyr, or Gln, but may be present or absent; N 18 is Phe, His, Tyr, or Thr.

[0015] In one embodiment, the alpha chain variable domain further comprises CDR1 and CDR2, wherein CDR1 comprises any one of the alpha chain variable domain CDR1 amino acid sequences shown in Table 2, and CDR2 independently comprises any one of the alpha chain variable domain CDR2 amino acid sequences shown in Table 2.

[0016] In another embodiment, the beta chain variable domain further comprises CDR1 and CDR2, wherein CDR1 comprises any one of the beta chain variable CDR1 amino acid sequences shown in Table 2, and CDR2 independently comprises any one of the beta chain variable domain CDR2 amino acid sequences shown in Table 2.

[0017] The TCR may comprise at least one TCR alpha chain variable domain and / or at least one beta chain variable domain, or the TCR may comprise a TCR alpha chain variable domain and a TCR beta chain variable domain.

[0018] In one embodiment, the TCR comprises an alpha chain variable domain CDR1, CDR2, and CDR3 comprised within any one of the alpha chain variable domain sequences listed in Table 4, and a beta chain variable domain CDR1, CDR2, and CDR3 comprised within any one of the beta chain variable domain sequences listed in Table 4.

[0019] In another embodiment, the TCR comprises an alpha chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the alpha chain variable domain amino acid sequences listed in Table 4.

[0020] In yet another embodiment, the TCR comprises a beta chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 4.

[0021] In one embodiment, the TCR comprises (a) an alpha chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the alpha chain variable domain amino acid sequences listed in Table 4, and (b) a beta chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 4.

[0022] In one embodiment, the TCR comprises (a) an alpha chain variable domain CD1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337, 353, 369, 385, 401, 417, 433, 449, 465, 481, 497, 513, 529, 545, 561, 577, and 593 an R1 domain; and (b) an alpha chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 466, 482, 498, 514, 530, 546, 562, 578, and 594. (c) an alpha chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307, 323, 339, 355, 371, 387, 403, 419, 435, 451, 467, 483, 499, 515, 531, 547, 563, 579, and 595; and (d) SEQ ID NO: (e) a beta chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 110, 113, 114, 115, 116, 117, 118, 120, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 140, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 160, 161, 162, 163, 164, 165, 166, 170, 171, 172, 173, 174,(f) a beta chain variable domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, 14, 15, 17, 18, 20, 21, 23, 25, 26, 28, 29, 31, 33, 34, 36, 37, 39, 41, 42, 44, 45, 47, 49, 50, 52, 53, 55, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554, 570, 586, and 602; and a beta chain variable domain CDR3 having an amino acid sequence selected from the group consisting of: 7, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379, 395, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555, 571, 587, and 603.

[0023] In one embodiment, the TCR is selected from the group consisting of SEQ ID NOs: 7 / 15, 23 / 31, 39 / 47, 55 / 63, 71 / 79, 87 / 95, 103 / 111, 119 / 127, 135 / 143, 151 / 159, 167 / 175, 183 / 191, 199 / 207, 215 / 223, 231 / 239, 247 / 255, 263 / 271, 279 / 287, 295 / 303, 311 / 319, 327 / 335, 343 / and 599 / 607.

[0024] In one embodiment, the TCR is selected from the group consisting of SEQ ID NOs: 7 / 15, 23 / 31, 39 / 47, 55 / 63, 71 / 79, 87 / 95, 103 / 111, 119 / 127, 135 / 143, 151 / 159, 167 / 175, 183 / 191, 199 / 207, 215 / 223, 231 / 239, 247 / 255, 263 / 271, 279 / 287, 295 / 303, 311 / 319, 327 / 335, 343 / 351, 35 The CDR sequences comprised within an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of: 9 / 367, 375 / 383, 391 / 399, 407 / 415, 423 / 431, 439 / 447, 455 / 463, 471 / 479, 487 / 495, 503 / 511, 519 / 527, 535 / 543, 551 / 559, 567 / 575, 583 / 591, and 599 / 607.

[0025] In another embodiment, the TCR is selected from the group consisting of SEQ ID NOs: 87 / 31, 23 / 95, 231 / 607, 231 / 223, 231 / 591, 231 / 255, 231 / 271, 231 / 79, 231 / 47, 231 / 399, 599 / 239, 599 / 223, 599 / 591, 599 / 255, 599 / 271, 599 / 79, 599 / 47, 599 / 399, 215 / 239, 215 / 607, 215 / 591, 215 / 255, 215 / 271, 215 / 79, 215 / 47, 215 / 399, 583 / 239, 583 / 607, 583 / 223, 583 / 255 , 583 / 271, 583 / 79, 583 / 47, 583 / 399, 247 / 239, 247 / 607, 247 / 223, 247 / 591, 247 / 271, 247 / 79, 247 / 47, 247 / 399, 263 / 239, 263 / 607, 263 / 223, 263 / 591, 263 / 255, 263 / 79, 263 / 47, 263 / 399, 71 / 239, 71 / 607, 71 / 223, 71 / 591, 71 / 255, 71 / 271, 71 / 47, 71 / 399, 39 / 239, 39 / 607, 39 / 223, 39 / 591, 39 / 255, 39 / 27 1, 39 / 79, 39 / 399, 391 / 239, 391 / 607, 391 / 223, 391 / 591, 391 / 255, 391 / 271, 391 / 79, 391 / 47, 439 / 127, 439 / 319, 439 / 287, 439 / 15, 439 / 111, 439 / 383, 439 / 191, 439 / 511, 439 / 527, 439 / 559, 439 / 207, 119 / 447, 119 / 319, 119 / 287, 119 / 15, 119 / 111, 119 / 383, 119 / 191, 119 / 511, 119 / 527, 119 / 559, 119 / 20 7, 311 / 447, 311 / 127, 311 / 287, 311 / 15, 311 / 111, 311 / 383, 311 / 191, 311 / 511, 311 / 527, 311 / 559, 311 / 207, 279 / 447, 279 / 127, 279 / 319, 279 / 15, 279 / 1 11, 279 / 383, 279 / 191, 279 / 511, 279 / 527, 279 / 559, 279 / 207, 7 / 447, 7 / 127, 7 / 319, 7 / 287, 7 / 111, 7 / 383, 7 / 191, 7 / 511, 7 / 527, 7 / 559, 7 / 207, 103 / 447,103 / 127, 103 / 319, 103 / 287, 103 / 15, 103 / 383, 103 / 191, 103 / 511, 103 / 527, 103 / 559, 103 / 207, 375 / 447, 375 / 127, 375 / 319, 375 / 287, 375 / 15, 375 / 111, 375 / 191, 375 / 511, 375 / 527, 375 / 559, 375 / 207, 183 / 447, 183 / 127, 183 / 319, 183 / 287, 183 / 15, 183 / 111, 183 / 383, 183 / 511, 183 / 527, 183 / 559, 183 / 207, 503 / 447, 503 / 127, 503 / 319, 503 / 287, 503 / 15, 503 / 111, 503 / 383, 503 / 191, 503 / 527, 503 / 559, 503 / 207, 519 / 447, 519 / 127, 519 / 319, 519 / 287, 519 / 15, 519 / 111, 519 / 383, 519 / 191, 519 / 511, 519 / 559, 519 / 207, 551 / 447, 551 / 127, 551 / 319, 551 / 287, 551 / 15, 551 / 111, 551 / 383, 551 / The alpha chain variable domain / beta chain variable domain amino acid sequence pair is selected from the group consisting of: 191, 551 / 511, 551 / 527, 551 / 207, 199 / 447, 199 / 127, 199 / 319, 199 / 287, 199 / 15, 199 / 111, 199 / 383, 199 / 191, 199 / 511, 199 / 527, and 199 / 559.

[0026] In another embodiment, the TCR is selected from the group consisting of SEQ ID NOs: 87 / 31, 23 / 95, 231 / 607, 231 / 223, 231 / 591, 231 / 255, 231 / 271, 231 / 79, 231 / 47, 231 / 399, 599 / 239, 599 / 223, 599 / 591, 599 / 255, 599 / 271, 599 / 79, 599 / 47, 599 / 399, 215 / 239, 215 / 607, 215 / 591, 215 / 255, 215 / 271, 215 / 79, 215 / 47, 215 / 399, 583 / 239, 583 / 607, 583 / 223, 583 / 255 , 583 / 271, 583 / 79, 583 / 47, 583 / 399, 247 / 239, 247 / 607, 247 / 223, 247 / 591, 247 / 271, 247 / 79, 247 / 47, 247 / 399, 263 / 239, 263 / 607, 263 / 223, 263 / 591, 263 / 255, 263 / 79, 263 / 47, 263 / 399, 71 / 239, 71 / 607, 71 / 223, 71 / 591, 71 / 255, 71 / 271, 71 / 47, 71 / 399, 39 / 239, 39 / 607, 39 / 223, 39 / 591, 39 / 255, 39 / 27 1, 39 / 79, 39 / 399, 391 / 239, 391 / 607, 391 / 223, 391 / 591, 391 / 255, 391 / 271, 391 / 79, 391 / 47, 439 / 127, 439 / 319, 439 / 287, 439 / 15, 439 / 111, 439 / 383, 439 / 191, 439 / 511, 439 / 527, 439 / 559, 439 / 207, 119 / 447, 119 / 319, 119 / 287, 119 / 15, 119 / 111, 119 / 383, 119 / 191, 119 / 511, 119 / 527, 119 / 559, 119 / 20 7, 311 / 447, 311 / 127, 311 / 287, 311 / 15, 311 / 111, 311 / 383, 311 / 191, 311 / 511, 311 / 527, 311 / 559, 311 / 207, 279 / 447, 279 / 127, 279 / 319, 279 / 15, 279 / 1 11, 279 / 383, 279 / 191, 279 / 511, 279 / 527, 279 / 559, 279 / 207, 7 / 447, 7 / 127, 7 / 319, 7 / 287, 7 / 111, 7 / 383, 7 / 191, 7 / 511, 7 / 527, 7 / 559, 7 / 207, 103 / 447,103 / 127, 103 / 319, 103 / 287, 103 / 15, 103 / 383, 103 / 191, 103 / 511, 103 / 527, 103 / 559, 103 / 207, 375 / 447, 375 / 127, 375 / 319, 375 / 287, 375 / 15, 375 / 111, 375 / 191, 375 / 511, 375 / 527, 375 / 559, 375 / 20 7, 183 / 447, 183 / 127, 183 / 319, 183 / 287, 183 / 15, 183 / 111, 183 / 383, 183 / 511, 183 / 527, 183 / 559, 183 / 207, 503 / 447, 503 / 127, 503 / 319, 503 / 287, 503 / 15, 503 / 111, 503 / 383, 503 / 191, 503 / 527, 503 / 5 59, 503 / 207, 519 / 447, 519 / 127, 519 / 319, 519 / 287, 519 / 15, 519 / 111, 519 / 383, 519 / 191, 519 / 511, 519 / 559, 519 / 207, 551 / 447, 551 / 127, 551 / 319, 551 / 287, 551 / 15, 551 / 111, 551 / 383, 551 / 191, 551 / The CDR sequences comprised within an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of: 511, 551 / 527, 551 / 207, 199 / 447, 199 / 127, 199 / 319, 199 / 287, 199 / 15, 199 / 111, 199 / 383, 199 / 191, 199 / 511, 199 / 527, and 199 / 559.

[0027] In some embodiments, the invention provides TCRs comprising CDRs contained within an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of SEQ ID NOs: 668 / 676, 103 / 111, 439 / 447, and 503 / 511. In some embodiments, the TCRs of the disclosure comprise alpha chain variable domain complementarity determining regions (CDRs) CDR1, CDR2, and CDR3, and beta chain variable domain CDRs, CDR1, CDR2, and CDR3, comprising the amino acid sequences of a) SEQ ID NOs: 662, 663, 664, 670, 671, and 672, b) SEQ ID NOs: 97, 98, 99, 105, 106, and 107, c) SEQ ID NOs: 433, 434, 435, 441, 442, and 443, and d) SEQ ID NOs: 497, 498, 499, 505, 506, and 507, respectively.

[0028] The invention also provides TCRs (eg, isolated TCRs, or TCRs expressed in isolated cells) that complete binding with any one or more of the TCRs of the invention.

[0029] In some embodiments, the TCRs of the present invention further comprise a detectable moiety.

[0030] The present invention further provides pharmaceutical compositions comprising any of the TCRs of the present invention and a pharmaceutically acceptable carrier or diluent, as well as isolated cells displaying any of the TCRs of the present invention.

[0031] In one aspect, the invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding the alpha chain variable domain of any of the TCRs of the invention.

[0032] In another aspect, the present invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding the beta chain variable domain of any of the TCRs of the present invention.

[0033] The present invention also provides vectors comprising the polynucleotide molecules of the present invention, and cells expressing the vectors of the present invention.

[0034] In one aspect, the present invention provides a method of treating a subject having a MAGE-A4-related disease or disorder, the method comprising administering to the subject a therapeutically effective amount of a TCR (e.g., an isolated TCR, or a TCR expressed on an isolated cell), pharmaceutical composition, or a plurality of cells of the invention, thereby treating the subject.

[0035] In one embodiment, the MAGE-A4 associated disease or disorder is a MAGE-A4 associated cancer.

[0036] In one embodiment, the MAGE-A4 associated cancer is liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, metastatic solid tumor, colon cancer, stomach cancer, gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, or recurrent non-small cell lung cancer.

[0037] In some embodiments of the invention, a TCR (e.g., an isolated TCR, or a TCR expressed in an isolated cell), pharmaceutical composition, or a plurality of cells of the invention is administered to a subject in combination with a second therapeutic agent.

[0038] The TCR, pharmaceutical composition, or plurality of cells may be administered to a subject subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.

[0039] In one aspect, the present invention provides an isolated nucleic acid molecule encoding a T cell receptor (TCR), wherein the TCR specifically binds to an HLA-A2-presented cancer-testis antigen melanoma associated antigen 4 (MAGE-A4) peptide comprising the amino acid sequence of KVLEHVVRV (SEQ ID NO: 609) (MAGE-A4 286-294), and wherein the TCR has a property selected from the group consisting of: (a) not binding to cells expressing predicted off-target peptides, as determined by a luminescent assay; (b) activating T cell responses to approximately two-fold greater than patient-derived MAGE-A4-specific TCRs, as determined by a TCR-mediated T cell signaling luminescent bioassay; and (c) activating T cell responses to approximately two-fold greater than affinity-matured (e.g., by phage display) MAGE-A4-specific TCRs, as determined by a TCR-mediated T cell signaling luminescent bioassay.

[0040] In one embodiment, the isolated nucleic acid molecule encodes at least one TCR alpha chain variable domain and / or at least one beta chain variable domain.

[0041] In one embodiment, the TCR comprises an alpha chain variable domain complementarity determining region (CDR) 1, CDR2, and CDR3 comprised within any one of the alpha chain variable domain sequences listed in Table 4, and a beta chain variable domain CDR1, CDR2, and CDR3 comprised within any one of the beta chain variable domain sequences listed in Table 4.

[0042] In another embodiment, the TCR (e.g., an isolated TCR or a TCR expressed in an isolated cell) comprises an alpha chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the alpha chain variable domain amino acid sequences listed in Table 4.

[0043] In yet another embodiment, the TCR comprises a beta chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 4.

[0044] In one embodiment, the TCR comprises (a) an alpha chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the alpha chain variable domain amino acid sequences listed in Table 4, and (b) a beta chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 4.

[0045] In one embodiment, the isolated antigen binding protein comprises (a) an alpha chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337, 353, 369, 385, 401, 417, 433, 449, 465, 481, 497, 513, 529, 545, 561, 577, and 593; and (b) an alpha chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, and (c) an alpha chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of: 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 466, 482, 498, 514, 530, 546, 562, 578, and 594; and (d) an alpha chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307, 32 3, 339, 355, 371, 387, 403, 419, 435, 451, 467, 483, 499, 515, 531, 547, 563, 579, and 595; and (d) an alpha chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, 329, 345, 361, 377, 393, 409, 425, 441, 457, 473, 489, 505, 521, 537. , 553, 569, 585, and 601; and (e) a beta chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554, 570, 586, and 602;(f) a beta chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379, 395, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555, 571, 587, and 603.

[0046] In one embodiment, the TCR is selected from the group consisting of SEQ ID NOs: 7 / 15, 23 / 31, 39 / 47, 55 / 63, 71 / 79, 87 / 95, 103 / 111, 119 / 127, 135 / 143, 151 / 159, 167 / 175, 183 / 191, 199 / 207, 215 / 223, 231 / 239, 247 / 255, 263 / 271, 279 / 287, 295 / 303, 311 / 319, 327 / 335, 343 / and 599 / 607.

[0047] In another embodiment, the TCR is selected from the group consisting of SEQ ID NOs: 87 / 31, 23 / 95, 231 / 607, 231 / 223, 231 / 591, 231 / 255, 231 / 271, 231 / 79, 231 / 47, 231 / 399, 599 / 239, 599 / 223, 599 / 591, 599 / 255, 599 / 271, 599 / 79, 599 / 47, 599 / 399, 215 / 239, 215 / 607, 215 / 591, 215 / 255, 215 / 271, 215 / 79, 215 / 47, 215 / 399, 583 / 239, 583 / 607, 583 / 223, 583 / 255 , 583 / 271, 583 / 79, 583 / 47, 583 / 399, 247 / 239, 247 / 607, 247 / 223, 247 / 591, 247 / 271, 247 / 79, 247 / 47, 247 / 399, 263 / 239, 263 / 607, 263 / 223, 263 / 591, 263 / 255, 263 / 79, 263 / 47, 263 / 399, 71 / 239, 71 / 607, 71 / 223, 71 / 591, 71 / 255, 71 / 271, 71 / 47, 71 / 399, 39 / 239, 39 / 607, 39 / 223, 39 / 591, 39 / 255, 39 / 27 1, 39 / 79, 39 / 399, 391 / 239, 391 / 607, 391 / 223, 391 / 591, 391 / 255, 391 / 271, 391 / 79, 391 / 47, 439 / 127, 439 / 319, 439 / 287, 439 / 15, 439 / 111, 439 / 383, 439 / 191, 439 / 511, 439 / 527, 439 / 559, 439 / 207, 119 / 447, 119 / 319, 119 / 287, 119 / 15, 119 / 111, 119 / 383, 119 / 191, 119 / 511, 119 / 527, 119 / 559, 119 / 20 7, 311 / 447, 311 / 127, 311 / 287, 311 / 15, 311 / 111, 311 / 383, 311 / 191, 311 / 511, 311 / 527, 311 / 559, 311 / 207, 279 / 447, 279 / 127, 279 / 319, 279 / 15, 279 / 1 11, 279 / 383, 279 / 191, 279 / 511, 279 / 527, 279 / 559, 279 / 207, 7 / 447, 7 / 127, 7 / 319, 7 / 287, 7 / 111, 7 / 383, 7 / 191, 7 / 511, 7 / 527, 7 / 559, 7 / 207, 103 / 447,103 / 127, 103 / 319, 103 / 287, 103 / 15, 103 / 383, 103 / 191, 103 / 511, 103 / 527, 103 / 559, 103 / 207, 375 / 447, 375 / 127, 375 / 319, 375 / 287, 375 / 15, 375 / 111, 375 / 191, 375 / 511, 375 / 527, 375 / 559, 375 / 207, 183 / 447, 183 / 127, 183 / 319, 183 / 287, 183 / 15, 183 / 111, 183 / 383, 183 / 511, 183 / 527, 183 / 559, 183 / 207, 503 / 447, 503 / 127, 503 / 319, 503 / 287, 503 / 15, 503 / 111, 503 / 383, 503 / 191, 503 / 527, 503 / 559, 503 / 207, 519 / 447, 519 / 127, 519 / 319, 519 / 287, 519 / 15, 519 / 111, 519 / 383, 519 / 191, 519 / 511, 510 / 559, 519 / 207, 551 / 447, 551 / 127, 551 / 319, 551 / 287, 551 / 15, 551 / 111, 551 / 383, 551 / The alpha chain variable domain / beta chain variable domain amino acid sequence pair is selected from the group consisting of: 191, 551 / 511, 551 / 527, 551 / 207, 199 / 447, 199 / 127, 199 / 319, 199 / 287, 199 / 15, 199 / 111, 199 / 383, 199 / 191, 199 / 511, 199 / 527, and 199 / 559.

[0048] In one embodiment, the isolated antigen binding protein comprises (a) an alpha chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452, 468, 484, 500, 516, 532, 548, 564, 580, and 596; and (b) an alpha chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5, 21, 37, 53 , 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261, 277, 293, 309, 325, 341, 357, 373, 389, 405, 421, 437, 453, 469, 485, 501, 517, 533, 549, 565, 581, and 597; and (c) an alpha chain variable domain CDR2 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 245, 261, 277, 293, 309, 325, 341, 357, 373, 389, 405, 421, 437, 453, 469, 485, 501, 517, 533, 549, 565, 581, and 597. and (d) an alpha chain variable domain CDR3 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, (e) a beta chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285, 301, 317, 333, 349, 365, 381, 397, 413, 429, 445, 461, 477, 493, 509, 525, 541, 557, 573, 589,and 605; and (f) a beta chain variable domain CDR3 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, ​​398, 414, 430, 446, 462, 478, 494, 510, 526, 542, 558, 574, 590, and 606.

[0049] In one embodiment, the TCR is selected from the group consisting of SEQ ID NOs: 8 / 16, 24 / 32, 40 / 48, 56 / 64, 72 / 80, 88 / 96, 104 / 112, 120 / 128, 136 / 144, 152 / 160, 168 / 176, 184 / 192, 200 / 208, 216 / 224, 232 / 240, 248 / 256, 264 / 272, 280 / 288, 296 / 304, 312 / 320, 328 / 336, 34 4 / 352, 360 / 368, 376 / 384, 392 / 400, 408 / 416, 424 / 432, 440 / 448, 456 / 464, 472 / 480, 488 / 496, 504 / 512, 520 / 528, 536 / 544, 552 / 560, 568 / 576, 584 / 592, 600 / 608.

[0050] The present invention also provides a vector comprising an isolated nucleic acid molecule of the present invention and an isolated cell comprising a vector of the present invention.

[0051] In one aspect, the present invention provides a method of treating a subject having a MAGE-A4 related disease or disorder, comprising administering to the subject a plurality of cells according to claim 42, thereby treating the subject.

[0052] In one embodiment, the MAGE-A4 associated disease or disorder is a MAGE-A4 associated cancer.

[0053] In one embodiment, the MAGE-A4 associated cancer is liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, metastatic solid tumor, colon cancer, stomach cancer, gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, or recurrent non-small cell lung cancer.

[0054] In one embodiment, the plurality of cells is administered to the subject in combination with a second therapeutic agent.

[0055] In one aspect, the present invention provides KVLEHVVRV (SEQ ID NO: 609) (MAGE-A4 The present invention provides a T cell receptor (TCR) (e.g., an isolated TCR, or a TCR expressed on isolated cells) that specifically binds to an HLA-A2-presented cancer-testis antigen melanoma-associated antigen 4 (MAGE-A4) peptide comprising an amino acid sequence of amino acids 286-294, wherein the TCR has a property selected from the group consisting of: (a) not binding to cells expressing predicted off-target peptides, as determined by a luminescence assay; (b) activating a T cell response with a greater signal-to-noise ratio than a patient-derived MAGE-A4-specific TCR, as determined by a TCR-mediated T cell signaling luminescence bioassay; and (c) activating a T cell response approximately two-fold greater than an affinity-matured (e.g., phage-displayed) MAGE-A4-specific TCR, as determined by a TCR-mediated T cell signaling luminescence bioassay.

[0056] In some embodiments, the TCR activates T cell responses about 2-fold greater, or about 3-fold greater, or about 4-fold greater than patient-derived MAGE-A4-specific TCRs as determined by a TCR-mediated T cell signaling luminescent bioassay. In some embodiments, the TCR activates T cell responses about 2-fold greater, or about 3-fold greater, or about 4-fold greater than affinity-matured (e.g., by phage display) MAGE-A4-specific TCRs as determined by a TCR-mediated T cell signaling luminescent bioassay.

[0057] In one aspect, the disclosure provides a T cell receptor (TCR) (e.g., an isolated TCR, or a TCR expressed by an isolated cell) that specifically binds to an HLA-A2-presented cancer-testis antigen melanoma associated antigen 4 (MAGE-A4) peptide comprising the amino acid sequence of GVYDGREHTV (SEQ ID NO: 612) (MAGE-A4 230-239), wherein the TCR comprises a complementarity-determining region 3 (CDR3) comprised in conjunction with an alpha chain variable domain of any one of SEQ ID NOs: 620, 636, 652, 668, 684, 700, 716, 732, 748, 764, 780, 796, 812, 828, 844, and 860.

[0058] In one aspect, the disclosure provides a T cell receptor (TCR) (e.g., an isolated TCR, or a TCR expressed by an isolated cell) that specifically binds to an HLA-A2-presented cancer-testis antigen melanoma associated antigen 4 (MAGE-A4) peptide comprising the amino acid sequence of GVYDGREHTV (SEQ ID NO: 612) (MAGE-A4 230-239), wherein the TCR comprises a complementarity-determining region 3 (CDR3) contained within the beta chain variable domain of any one of SEQ ID NOs: 628, 644, 660, 676, 692, 708, 724, 740, 756, 772, 788, 804, 820, 836, 852, and 868.

[0059] In some embodiments, the alpha chain variable domain further comprises CDR1 and CDR2, wherein CDR1 comprises any one of the alpha chain variable domain CDR1 amino acid sequences shown in Table 6, and CDR2 independently comprises any one of the alpha chain variable domain CDR2 amino acid sequences shown in Table 6. In some embodiments, the beta chain variable domain further comprises CDR1 and CDR2, wherein CDR1 comprises any one of the beta chain variable CDR1 amino acid sequences shown in Table 6, and CDR2 independently comprises any one of the beta chain variable domain CDR2 amino acid sequences shown in Table 6. In some embodiments, the TCR comprises at least one TCR alpha chain variable domain and / or at least one beta chain variable domain. In some embodiments, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain.

[0060] In some embodiments, the TCR comprises an alpha chain variable domain CDR1, CDR2, and CDR3 comprised within any one of the alpha chain variable domain sequences listed in Table 8, and a beta chain variable domain CDR1, CDR2, and CDR3 comprised within any one of the beta chain variable domain sequences listed in Table 8. In some embodiments, the TCR comprises an alpha chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 8. In some embodiments, the TCR comprises a beta chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta chain variable domain amino acid sequences listed in Table 8. In some embodiments, the TCR comprises (a) an alpha chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the alpha chain variable domain amino acid sequences listed in Table 8, and (b) a beta chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 8.

[0061] In some embodiments, the TCR comprises (a) an alpha chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 614, 630, 646, 662, 678, 694, 710, 726, 742, 758, 774, 790, 806, 822, 838, and 854; and (b) an alpha chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 615, 631, 647, 663, 679, 695, 711, 727, 74 (c) an alpha chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 616, 632, 648, 664, 680, 696, 712, 728, 744, 760, 776, 792, 808, 824, 840, and 856; (d) a beta chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 622, 638, 654, 670, 686, 702, 718, 734, 750, 766, 782, 798, 814, 830, 846, and 862; and (e) a beta chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 623, 639, 655, 671, 687, 703, 719, 735, 751, 767, 783. , 799, 815, 831, 847, and 863; and (f) a beta chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 624, 640, 656, 672, 688, 704, 720, 736, 752, 768, 784, 800, 816, 832, 848, and 864.

[0062] In some embodiments, the TCR comprises an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of SEQ ID NOs: 620 / 628, 636 / 644, 652 / 660, 668 / 676, 684 / 692, 700 / 708, 716 / 724, 732 / 740, 748 / 756, 764 / 772, 780 / 788, 796 / 804, 812 / 820, 828 / 836, 844 / 852, and 860 / 868. In some embodiments, the TCR is selected from the group consisting of SEQ ID NOs: 620 / 628, 620 / 644, 620 / 660, 620 / 676, 620 / 692, 620 / 708, 620 / 724, 620 / 740, 620 / 756, 620 / 772, 620 / 788, 620 / 804, 620 / 820, 620 / 836, 620 / 852, 620 / 868, 636 / 628, 636 / 644, 636 / 660, 636 / 676, 636 / 692, 636 / 708, 636 / 724, 636 / 740, 636 / 756, 636 / 772, 636 / 788, 636 / 804, 636 / 820, 636 / 836, 636 / 852, 636 / 868, 652 / 628, 652 / 644, 652 / 660, 652 / 676, 652 / 692, 652 / 708, 652 / 724, 652 / 740, 652 / 756, 652 / 772, 652 / 788, 652 / 804, 652 / 820, 652 / 836, 652 / 852, 652 / 868, 668 / 628, 668 / 644, 668 / 660, 668 / 676, 668 / 692, 668 / 708, 668 / 724, 668 / 740, 668 / 756, 668 / 772, 668 / 788, 668 / 804, 668 / 820, 668 / 836, 668 / 852, 668 / 868, 684 / 628, 684 / 644, 684 / 660, 684 / 676, 684 / 692, 684 / 708, 684 / 724, 684 / 740, 684 / 756, 684 / 772, 684 / 788, 684 / 804, 684 / 820, 684 / 836, 684 / 852, 684 / 868, 700 / 628, 700 / 644, 700 / 660, 700 / 676, 700 / 692, 700 / 708, 700 / 724, 700 / 740, 700 / 756, 700 / 772, 700 / 788, 700 / 804, 700 / 820, 700 / 836, 700 / 852, 700 / 868, 716 / 628,716 / 644、716 / 660、716 / 676、716 / 692、716 / 708、716 / 724、716 / 740、716 / 756、716 / 772、716 / 788、716 / 804、716 / 820、716 / 836、716 / 852、716 / 868、732 / 628、732 / 644、732 / 660、732 / 676、732 / 692、732 / 708、732 / 724、732 / 740、732 / 756、732 / 772、732 / 788、732 / 804、732 / 820、732 / 836、732 / 852、732 / 868、748 / 628、748 / 644、748 / 660、748 / 676、748 / 692、748 / 708、748 / 724、748 / 740、748 / 756、748 / 772、748 / 788、748 / 804、748 / 820、748 / 836、748 / 852、748 / 868、764 / 628、764 / 644、764 / 660、764 / 676、764 / 692、764 / 708、764 / 724、764 / 740、764 / 756、764 / 772、764 / 788、764 / 804、764 / 820、764 / 836、764 / 852、764 / 868、780 / 628、780 / 644、780 / 660、780 / 676、780 / 692、780 / 708、780 / 724、780 / 740、780 / 756、780 / 772、780 / 788、780 / 804、780 / 820、780 / 836、780 / 852、780 / 868、796 / 628、796 / 644、796 / 660、796 / 676、796 / 692、796 / 708、796 / 724、796 / 740、796 / 756、796 / 772、796 / 788、796 / 804、796 / 820、796 / 836、796 / 852、796 / 868、812 / 628、812 / 644、812 / 660、812 / 676、812 / 692、812 / 708、812 / 724、812 / 740、812 / 756、812 / 772、812 / 788、812 / 804、812 / 820、812 / 836、812 / 852、812 / 868、828 / 628、828 / 644、828 / 660、828 / 676、828 / 692、828 / 708、828 / 724、828 / 740、828 / 756、828 / 772、828 / 788、828 / 804、828 / 820、828 / 836、828 / 852, 828 / 868, 844 / 628, 844 / 644, 844 / 660, 844 / 676, 844 / 692, 844 / 708, 844 / 724, 844 / 740, 844 / 756, 844 / 772, 844 / 788, 844 / 804, 844 / 820, 844 / 836, 844 / 852, 844 / 868, 860 / 628, 860 / 644 , 860 / 660, 860 / 676, 860 / 692, 860 / 708, 860 / 724, 860 / 740, 860 / 756, 860 / 772, 860 / 788, 860 / 804, 860 / 820, 860 / 836, 860 / 852, and 860 / 868.

[0063] In some embodiments, the TCR comprises a detectable moiety. In some embodiments, the TCR has an on-target binding / off-target binding value of greater than 2.5, greater than 3, greater than 3.5, greater than 4, greater than 4.5, greater than 5, greater than 10, greater than 15, greater than 20, greater than 50, greater than 100, greater than 200, greater than 300, greater than 400, greater than 500, greater than 600, greater than 700, greater than 800, greater than 900, or greater than 1000. In some embodiments, the TCR has an on-target binding / off-target binding value of greater than 10. In some embodiments, the TCR has an on-target binding / off-target binding value of greater than 500. In some embodiments, the TCR competes for binding to a TCR described herein (e.g., an isolated TCR or a TCR expressed in an isolated cell).

[0064] In one aspect, the present disclosure provides a pharmaceutical composition comprising a TCR described herein (e.g., an isolated TCR, or a TCR expressed in an isolated cell) and a pharmaceutically acceptable carrier or diluent. In one aspect, the present disclosure provides an isolated cell presenting a TCR described herein. In one aspect, the present disclosure provides an isolated polynucleic acid comprising a polynucleotide sequence encoding an alpha chain variable domain of a TCR described herein (e.g., an isolated TCR, or a TCR expressed in an isolated cell). In one aspect, the present disclosure provides an isolated polynucleic acid comprising a polynucleotide sequence encoding a beta chain variable domain of a TCR described herein (e.g., an isolated TCR, or a TCR expressed in an isolated cell). In one aspect, the present disclosure provides a vector comprising a polynucleotide sequence described herein. In one aspect, the present disclosure provides an isolated cell expressing the vector.

[0065] In one aspect, the disclosure provides a method of treating a subject having a MAGE-A4-associated disease or disorder, comprising administering to the subject a therapeutically effective amount of a TCR described herein (e.g., an isolated TCR, or a TCR expressed on an isolated cell), a pharmaceutical composition described herein, or an isolated cell described herein, thereby treating the subject. In some embodiments, the MAGE-A4-associated disease or disorder is a MAGE-A4-associated cancer. In some embodiments, the MAGE-A4 associated cancer is liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, metastatic solid tumor, colon cancer, stomach cancer, gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, or recurrent non-small cell lung cancer. In some embodiments, the TCR, pharmaceutical composition, or cell is administered to a subject in combination with a second therapeutic agent. In some embodiments, administration is parenteral.

[0066] In one aspect, the disclosure provides an isolated nucleic acid molecule encoding a T cell receptor (TCR), wherein the TCR specifically binds to an HLA-A2-presented cancer-testis antigen melanoma associated antigen 4 (MAGE-A4) peptide comprising the amino acid sequence of GVYDGREHTV (SEQ ID NO: 612) (MAGE-A4 230-239), wherein the TCR has a property selected from the group consisting of: (a) not binding to cells expressing predicted off-target peptides, as determined by a luminescence assay; (b) not binding to cells expressing predicted off-target peptides, as determined by a flow cytometry assay; (c) activating T cell responses to approximately two-fold greater than a patient-derived MAGE-A4-specific TCR, as determined by a TCR-mediated T cell signaling luminescence bioassay; and (d) activating T cell responses to approximately two-fold greater than an affinity-matured (e.g., by phage display) MAGE-A4-specific TCR, as determined by a TCR-mediated T cell signaling luminescence bioassay. In some embodiments, the isolated nucleic acid molecule encodes at least one TCR alpha chain variable domain and / or at least one beta chain variable domain.

[0067] In some embodiments, the TCR comprises an alpha chain variable domain complementarity determining region (CDR) 1, CDR2, and CDR3 comprised within any one of the alpha chain variable domain sequences listed in Table 8, and a beta chain variable domain CDR1, CDR2, and CDR3 comprised within any one of the beta chain variable domain sequences listed in Table 8. In some embodiments, the TCR comprises an alpha chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 8. In some embodiments, the TCR comprises a beta chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta chain variable domain amino acid sequences listed in Table 8.

[0068] In some embodiments, the TCR comprises (a) an alpha chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the alpha chain variable domain amino acid sequences listed in Table 8, and (b) a beta chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 8. In some embodiments, the TCR comprises (a) an alpha chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 614, 630, 646, 662, 678, 694, 710, 726, 742, 758, 774, 790, 806, 822, 838, and 854, and (b) an alpha chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 615, 631, 647, 663, 679, 695, 711, 727, 74 (c) an alpha chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 616, 632, 648, 664, 680, 696, 712, 728, 744, 760, 776, 792, 808, 824, 840, and 856; (d) a beta chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 622, 638, 654, 670, 686, 702, 718, 734, 750, 766, 782, 798, 814, 830, 846, and 862; and (e) a beta chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 623, 639, 655, 671, 687, 703, 719, 735, 751, 767, 783. , 799, 815, 831, 847, and 863; and (f) a beta chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 624, 640, 656, 672, 688, 704, 720, 736, 752, 768, 784, 800, 816, 832, 848, and 864.

[0069] In some embodiments, the TCR comprises an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of SEQ ID NOs: 620 / 628, 636 / 644, 652 / 660, 668 / 676, 684 / 692, 700 / 708, 716 / 724, 732 / 740, 748 / 756, 764 / 772, 780 / 788, 796 / 804, 812 / 820, 828 / 836, 844 / 852, and 860 / 868. In some embodiments, the TCR is selected from the group consisting of SEQ ID NOs: 620 / 628, 620 / 644, 620 / 660, 620 / 676, 620 / 692, 620 / 708, 620 / 724, 620 / 740, 620 / 756, 620 / 772, 620 / 788, 620 / 804, 620 / 820, 620 / 836, 620 / 852, 620 / 868, 636 / 628, 636 / 644, 636 / 660, 636 / 676, 636 / 692, 636 / 708, 636 / 724, 636 / 740, 636 / 756, 636 / 772, 636 / 788, 636 / 804, 636 / 820, 636 / 836, 636 / 852, 636 / 868, 652 / 628, 652 / 644, 652 / 660, 652 / 676, 652 / 692, 652 / 708, 652 / 724, 652 / 740, 652 / 756, 652 / 772, 652 / 788, 652 / 804, 652 / 820, 652 / 836, 652 / 852, 652 / 868, 668 / 628, 668 / 644, 668 / 660, 668 / 676, 668 / 692, 668 / 708, 668 / 724, 668 / 740, 668 / 756, 668 / 772, 668 / 788, 668 / 804, 668 / 820, 668 / 836, 668 / 852, 668 / 868, 684 / 628, 684 / 644, 684 / 660, 684 / 676, 684 / 692, 684 / 708, 684 / 724, 684 / 740, 684 / 756, 684 / 772, 684 / 788, 684 / 804, 684 / 820, 684 / 836, 684 / 852, 684 / 868, 700 / 628, 700 / 644, 700 / 660, 700 / 676, 700 / 692, 700 / 708, 700 / 724, 700 / 740, 700 / 756, 700 / 772, 700 / 788, 700 / 804, 700 / 820, 700 / 836, 700 / 852, 700 / 868, 716 / 628,716 / 644、716 / 660、716 / 676、716 / 692、716 / 708、716 / 724、716 / 740、716 / 756、716 / 772、716 / 788、716 / 804、716 / 820、716 / 836、716 / 852、716 / 868、732 / 628、732 / 644、732 / 660、732 / 676、732 / 692、732 / 708、732 / 724、732 / 740、732 / 756、732 / 772、732 / 788、732 / 804、732 / 820、732 / 836、732 / 852、732 / 868、748 / 628、748 / 644、748 / 660、748 / 676、748 / 692、748 / 708、748 / 724、748 / 740、748 / 756、748 / 772、748 / 788、748 / 804、748 / 820、748 / 836、748 / 852、748 / 868、764 / 628、764 / 644、764 / 660、764 / 676、764 / 692、764 / 708、764 / 724、764 / 740、764 / 756、764 / 772、764 / 788、764 / 804、764 / 820、764 / 836、764 / 852、764 / 868、780 / 628、780 / 644、780 / 660、780 / 676、780 / 692、780 / 708、780 / 724、780 / 740、780 / 756、780 / 772、780 / 788、780 / 804、780 / 820、780 / 836、780 / 852、780 / 868、796 / 628、796 / 644、796 / 660、796 / 676、796 / 692、796 / 708、796 / 724、796 / 740、796 / 756、796 / 772、796 / 788、796 / 804、796 / 820、796 / 836、796 / 852、796 / 868、812 / 628、812 / 644、812 / 660、812 / 676、812 / 692、812 / 708、812 / 724、812 / 740、812 / 756、812 / 772、812 / 788、812 / 804、812 / 820、812 / 836、812 / 852、812 / 868、828 / 628、828 / 644、828 / 660、828 / 676、828 / 692、828 / 708、828 / 724、828 / 740、828 / 756、828 / 772、828 / 788、828 / 804、828 / 820、828 / 836、828 / 852, 828 / 868, 844 / 628, 844 / 644, 844 / 660, 844 / 676, 844 / 692, 844 / 708, 844 / 724, 844 / 740, 844 / 756, 844 / 772, 844 / 788, 844 / 804, 844 / 820, 844 / 836, 844 / 852, 844 / 868, 860 / 628, 860 / 644 , 860 / 660, 860 / 676, 860 / 692, 860 / 708, 860 / 724, 860 / 740, 860 / 756, 860 / 772, 860 / 788, 860 / 804, 860 / 820, 860 / 836, 860 / 852, and 860 / 868.

[0070] In some embodiments, the TCR comprises (a) an alpha chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 617, 633, 649, 665, 681, 697, 713, 729, 745, 761, 777, 793, 809, 825, 841, and 857; and (b) an alpha chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 618, 634, 650, 666, 682, 698, 714, 730, 746, 777, 793, 809, 825, 841, and 857. (c) an alpha chain variable domain CDR2 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 619, 635, 651, 667, 683, 699, 715, 731, 747, 763, 779, 795, 811, 827, 843, and 859. (d) a main CDR3; and (e) a beta chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 625, 641, 657, 673, 689, 705, 721, 737, 753, 769, 785, 801, 817, 833, 849, and 865; and (f) a beta chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 626, 642, 658, 674, 690, 706, 722, 738, 754, 770, 786, 802, 818, 834, 850, and 866; and (f) a beta chain variable domain CDR3 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 627, 643, 659, 675, 691, 707, 723, 739, 755, 771, 787, 803, 819, 835, 851, and 867. In some embodiments, the TCR comprises an alpha chain variable domain / beta chain variable domain nucleic acid sequence pair selected from the group consisting of SEQ ID NOs: 621 / 629, 637 / 645, 653 / 661, 669 / 677, 685 / 693, 701 / 709, 717 / 725, 733 / 741, 749 / 757, 765 / 773, 781 / 789, 797 / 805, 813 / 821, 829 / 837, 845 / 853, and 861 / 869.

[0071] In one aspect, the present disclosure provides a vector comprising the polynucleotide sequence of the isolated nucleic acid molecule described herein. In one aspect, the present disclosure provides an isolated cell comprising the vector. In one aspect, the present disclosure provides a method of treating a subject having a MAGE-A4-associated disease or disorder, comprising administering the cell to the subject, thereby treating the subject. In some embodiments, the MAGE-A4-associated disease or disorder is a MAGE-A4-associated cancer. In some embodiments, the MAGE-A4 associated cancer is liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, metastatic solid tumor, colon cancer, stomach cancer, gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, or recurrent non-small cell lung cancer. In some embodiments, the cells are administered to a subject in combination with a second therapeutic agent.

[0072] The present invention is further explained by the following detailed description and drawings. [Brief explanation of the drawings]

[0073] [Figure 1]FACS analysis shows the expression of TRAC-targeting MAGE-A4 TCRs in primary human T cells, quantified by pMHC tetramer analysis at day 14 postactivation. The MAGE-A4 target in these TCRs was either the MAGE-A4 (286-294) peptide (KVLEHVVRV, SEQ ID NO: 609, "KVLE") or the MAGE-A4 (230-239) peptide (GVYDGREHTV, SEQ ID NO: 612, "GVY"). The transgenic TCR sequences were introduced into primary human T cells by site-specific targeting of adeno-associated virus (AAV) vector insertion at the human TRAC locus. Cells were stained with the indicated peptide-MHC tetramer reagents (y-axis) and antibodies against surface antigen markers and analyzed by flow cytometry. The plot was gated on live, single CD8+ T cells. [Figure 2] Figure 2 shows the cytotoxic activity of MAGE-A4 TCR against MAGE-A4-expressing A375 melanoma cells (donor 1, 2-hour assay). Primary human T cells expressing TCRs against two different HLA-A2-restricted MAGE-A4-derived peptide antigens were tested for cytotoxic activity against HLA-A2*01 MAGE-A4-expressing A375 melanoma cells in a calcein AM dye-release assay. The MAGE-A4 target for these TCRs was either the MAGE-A4 (286-294) peptide (KVLEHVVRV, SEQ ID NO: 609, "KVLE") (Figure 2A) or the MAGE-A4 (230-239) peptide (GVYDGREHTV, SEQ ID NO: 612, "GVY") (Figure 2B). Control cells transduced with a TCR against an irrelevant antigen were included as a control for TCR target specificity. [Figure 3]Figure 3 shows the cytotoxic activity of MAGE-A4 TCRs against MAGE-A4-expressing A375 melanoma cells (donor 2, 2.5-hour assay). The MAGE-A4 target in these TCRs was either the MAGE-A4 (286-294) peptide (KVLEHVVRV, SEQ ID NO: 609, "KVLE") (Figure 3A) or the MAGE-A4 (230-239) peptide (GVYDGREHTV, SEQ ID NO: 612, "GVY") (Figure 3B). Primary human T cells expressing TCRs against two different HLA-A2-restricted MAGE-A4-derived peptide antigens were tested for cytotoxic activity against HLA-A2*01 MAGE-A4-expressing A375 melanoma cells in a calcein AM dye-release assay. Untransduced (UTD) T cells were included as a control for TCR target specificity. [Figure 4] TCR signaling measured by activation of AP1-RE-luciferase reporter activity is shown. [Figure 5] Shown is the mean + / - standard error of the mean (SEM) of interferon gamma signals from donors (n=3), demonstrating that the MAGE-A4(230-239)-specific TCR of the present disclosure exhibits specific activity against T2 cells pulsed with MAGE-A4(230-239) peptide. [Figure 6] Shown are the mean + / - standard error of the mean (SEM) of interferon gamma signals from donors (n=3) and demonstrate that the MAGE-A4(230-239)-specific TCR of the present disclosure exhibits specific activity against tumor cells expressing HLA-A2 and MAGE-A4. [Figure 7] Shown are the mean + / - standard error of the mean (SEM) of interferon gamma signals from donors (n=3) and demonstrate that the MAGE-A4(230-239)-specific TCR of the present disclosure exhibits specific activity against tumor cells expressing HLA-A2 and MAGE-A4. [Figure 8] Shown are the mean + / - standard error of the mean (SEM) of interferon-γ signals from donors (n=3) and demonstrate that the MAGE-A4(230-239)-specific TCR of the present disclosure exhibits specific activity even against HLA-A2-expressing tumor cells that express only low or moderate endogenous levels of MAGE-A4. DETAILED DESCRIPTION OF THE INVENTION

[0074] The present invention provides T cell receptors (TCRs) directed against the MAGE-A4 peptide antigen in the context of MHC (HLA-A2). The unique TCR sequences identified show specific binding to the small peptide MAGE-A4, which resides in the groove of the HLA molecule, and demonstrate T cell activation in reporter assays. Furthermore, the TCRs of the present invention do not cross-react with other "similar" peptides.

[0075] I. Definition In order that the present invention may be more readily understood, certain terms are first defined. Furthermore, it should be noted that whenever a value or range of values ​​for a parameter is listed, it is intended that values ​​and ranges intermediate to the listed values ​​are also part of the invention.

[0076] In the following description, for purposes of explanation, specific numbers, materials, and configurations are set forth to provide a thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention may be practiced without these specific details. In some instances, well-known features may be omitted or simplified so as not to obscure the present invention. Furthermore, references herein to phrases such as "one embodiment" or "embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Appearances of phrases such as "in one embodiment" in various places in this specification do not necessarily all refer to the same embodiment.

[0077] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0078] The terms "comprising" or "comprises" are used herein to refer to compositions, methods, and their respective components that are essential to the present disclosure, but may include elements not specified as essential.

[0079] The term "consisting of" refers to compositions, methods, and their respective components described herein, which exclude any element not recited in that description of an embodiment.

[0080] The term "T cell receptor" (TCR), as used herein, refers to a member of the immunoglobulin superfamily having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail, and is capable of specifically binding to an antigenic peptide bound to an MHC receptor (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). TCRs are found on the surface of cells and are generally composed of heterodimers having an α chain and a β chain (also known as TCRα and TCRβ, respectively), or a γ chain and a δ chain (also known as TCRγ and TCRδ, respectively). Similar to immunoglobulins, the extracellular portion of a TCR chain (e.g., α chain, β chain) comprises two immunoglobulin regions adjacent to the cell membrane: a variable region (e.g., the TCR variable α region or Vα and the TCR variable β region or Vβ, typically amino acids 1-116 according to Kabat numbering at the N-terminus), and one constant region (e.g., the TCR constant domain α or Cα, typically amino acids 117-259 according to Kabat numbering, and the TCR constant domain β or Cβ, typically amino acids 117-295 according to Kabat numbering). Also similar to immunoglobulins, the variable domains comprise complementarity-determining regions (CDRs) separated by framework regions (FRs). In certain embodiments, TCRs are found on the surface of T cells (or T lymphocytes) and associate with the CD3 complex. The source of the TCRs of the present disclosure may be from various animal species, such as human, mouse, rat, rabbit, or other mammals. In a preferred embodiment, the source of the TCR of the present invention is a mouse that has been genetically engineered to produce a TCR comprising a human alpha chain and a beta chain (see, e.g., PCT Publication No. WO2016 / 164492, the entire contents of which are incorporated herein by reference).

[0081] As used herein, the term "variable region" (variable region of the alpha chain (Vα) and variable region of the beta chain (Vβ)) refers to each of the alpha and beta chains that are directly involved in binding of the TCR to an antigen.

[0082] The "constant regions" of the alpha and beta chains are not directly involved in the binding of TCR to antigen, but exhibit various effector functions.

[0083] As used herein, the term "antigen" means any substance that causes the immune system to produce an antibody or specific cell-mediated immune response against it. A disease-associated antigen is any substance associated with any disease that causes the immune system to produce an antibody or specific cell-mediated response against it.

[0084] The term "MAGE-A4," "MAGEA4," or "melanoma-associated antigen A4" refers to a well-known cancer-testis antigen (CTA) that is re-expressed in multiple cancer types.

[0085] The nucleotide and amino acid sequences of full-length MAGE-A4 are provided in GenBank under accession number NM_001011548 (DNA sequence: SEQ ID NO:870, RNA sequence: SEQ ID NO:610, amino acid sequence: SEQ ID NO:611). The term "MAGE-A4" includes recombinant MAGE-A4 or a fragment thereof. The term also encompasses MAGE-A4 or a fragment thereof linked to a signal sequence, such as, for example, a histidine tag, mouse or human Fc, or ROR1. In certain embodiments, the term includes MAGE-A4 or a fragment thereof associated with, linked to, or presented by HLA-A2. As used herein, the numbering of specific MAGE-A4 amino acid residues within the full-length MAGE-A4 sequence is relative to SEQ ID NO:611.

[0086] The term "HLA" refers to the human leukocyte antigen (HLA) system or complex, a complex of genes that encodes major histocompatibility complex (MHC) proteins in humans. These cell surface proteins are involved in regulating the immune system in humans. HLA, corresponding to MHC class I (A, B, and C), present peptides from the interior of cells.

[0087] The term "HLA-A" refers to a group of human leukocyte antigens (HLA) encoded by the HLA-A locus. HLA-A is one of the three major types of human MHC class I cell surface receptors. The receptor is a heterodimer, composed of a heavy α chain and a smaller β chain. The α chain is encoded by the variant HLA-A gene, and the β chain (β2-microglobulin) is the invariant β2-microglobulin molecule.

[0088] The term "HLA-A2" (also referred to as "HLA-A2*01") refers to one of a group of specific class I major histocompatibility complex (MHC) alleles at the HLA-A locus, with the alpha chain encoded by the HLA-A*02 gene and the beta chain encoded by the beta2-microglobulin or B2M locus.

[0089] The terms "specifically binds to" or "specifically binds to" and the like mean that the TCR forms a complex with the antigen that is relatively stable under physiological conditions. Specific binding is at least about 1 x 10 -8 The binding can be characterized by an equilibrium dissociation constant equal to or less than M (e.g., a smaller KD indicates stronger binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, the TCRs of the present invention specifically bind to an HLA-A2-presented cancer-testis antigen melanoma-associated antigen A4 (MAGE-A4) peptide, for example, a peptide comprising amino acid residues 230-239 or 286-294 of MAGE-A4 (e.g., the full-length MAGE-A4 sequence of SEQ ID NO: 611).

[0090] The term "off-target peptide" refers to a peptide that differs from a target peptide (e.g., the MAGE-A4 230-239 peptide or the MAGE-A4 286-294 peptide) by one, two, three, four, five, or more amino acids. In certain embodiments, the term includes peptides that differ by three or fewer amino acids than the target peptide. For example, a 9-mer peptide is considered an "off-target" peptide if one, two, or three amino acids are not identical to the target peptide. In certain embodiments, amino acid identity is expressed in terms of "degree of similarity" (DoS). If six or more amino acids within a 9-mer peptide are identical, the DoS is 6. In certain embodiments, a peptide with a DoS ≤ 6 is considered an "off-target" peptide. The term "off-target" peptide also refers to a peptide that is similar to a target peptide based on sequence homology, predicted to bind to HLA-A2, and contained in a protein expressed in essential normal tissues.

[0091] The term "isolated" refers to a composition, compound, substance, or molecule that has been altered by human hands from its natural state. For example, a composition or substance that exists in nature is isolated if it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living animal is not isolated, but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is isolated as that term is used herein. More specifically, an isolated TCR can refer to a TCR that has been removed from a cell, e.g., a purified TCR. A TCR can also be expressed by an isolated cell, e.g., a cell that has been isolated from an animal or a cell from a cell culture. In this context, an isolated cell can express a TCR on its surface (i.e., the cell can "present" the TCR).

[0092] The term "recombinant," as used herein, refers to a TCR of the present invention that is made, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term refers to a TCR that is expressed in a non-human mammal (including a transgenic non-human mammal, e.g., a transgenic mouse) or cell (e.g., a CHO cell) expression system, or a TCR that is isolated from a recombinant combinatorial human antibody library.

[0093] As used herein, the terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to a polymeric form of nucleotides of any length. Polynucleotides can contain deoxyribonucleotides, ribonucleotides, and / or their analogs. Nucleotides can have any three-dimensional structure and perform any function, known or unknown. The term "polynucleotide" includes, for example, single-stranded, double-stranded, and triple-helical molecules, genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, antisense molecules, cDNA, recombinant polynucleotides, branched polynucleotides, aptamers, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can also include modified nucleic acid molecules (e.g., containing modified bases, sugars, and / or internucleotide linkers).

[0094] The term "polypeptide" is intended to refer to any polymer consisting essentially of any of the 20 naturally occurring amino acids, regardless of its size. The term "protein" is often used in reference to relatively large proteins, and "peptide" is often used in reference to small polypeptides, although the use of these terms often overlaps in the art. The term "polypeptide" generally refers to proteins, polypeptides, and peptides, unless otherwise specified. Generally, useful peptides according to the present disclosure generally range in size from about 0.1 to 100 KD or greater, or up to about 1000 KD, preferably between about 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30, and 50 KD, as determined by standard molecular sizing techniques such as centrifugation or SDS-polyacrylamide gel electrophoresis.

[0095] The term "vector" refers to a nucleic acid molecule that can replicate autonomously within a host cell and can accept foreign DNA. A vector carries its own origin of replication, one or more unique recognition sites for restriction endonucleases that can be used for the insertion of foreign DNA and usually selectable markers such as genes encoding antibiotic resistance, and often a recognition sequence (e.g., a promoter) for the expression of the inserted DNA. Common vectors include plasmid vectors and phage vectors.

[0096] In some embodiments, the TCRs of the present invention may be conjugated to a ligand, a detectable moiety, or a therapeutic moiety (an "immunoconjugate"), such as a cytotoxin, an anti-cancer drug, or any other therapeutic moiety that is useful for treating a disease or condition, including a MAGE-A4-associated disease or disorder, such as a MAGE-A4-associated cancer.

[0097] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detection of changes in protein concentration within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0098] The term "KD" refers to D or K d Also known as the equilibrium dissociation constant (KD), it is intended to refer to the equilibrium dissociation constant between a specific biomolecule and its binding partner. KD measurements are particularly useful for evaluating protein-protein interactions, such as antigen-binding protein-antigen interactions. The smaller the KD value, the greater (or stronger) the binding interaction or affinity between the antigen-binding protein and the antigen (e.g., target). The larger the KD value, the weaker the binding interaction or affinity between the antigen-binding protein and the antigen.

[0099] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity in at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm for sequence identity, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain cases, encode a polypeptide having the same or substantially the same amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0100] Sequence identity can be calculated using algorithms such as the Needleman-Wunsch algorithm for global alignment (Needleman and Wunsch 1970, J. Mol. Biol. 48:443-453) or the Smith-Waterman algorithm for local alignment (Smith and Waterman 1981, J. Mol. Biol. 147:195-197). Another preferred algorithm is that reported by Dufresne et al. in Nature Biotechnology (vol. 20, pp. 1269-71) in 2002 and implemented in the software GenePAST (GQ Life Sciences, Inc. Used in Boston, MA.

[0101] When applied to polypeptides, the term "substantial similarity" or "substantially similar" refers to two peptide sequences that share at least 90% sequence identity, and even more preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity, when optimally aligned, such as by programs like GAP or BESTFIT using default gap weighting. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of amino acid groups with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix published in Gonnet et al. (1992) Science 256:1443 45, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0102] Sequence similarity in polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software can determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutant protein, using default parameters. Programs such as GAP and BESTFIT are included. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). Sequences can also be compared using the Smith-Waterman homology search algorithm, using an affine gap search with a gap opening penalty of 12 and a gap extension penalty of 2, in the BLOSUM62 matrix. Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0103] A "patient-derived TCR" is a TCR generated by isolating the alpha and beta chains of a MAGE-A4-reactive TCR isolated from T lymphocytes that mediate in vivo tumor regression in a subject with a MAGE-A4-associated cancer.

[0104] An "affinity matured TCR" is a TCR generated by in vitro mutagenesis and selection. For example, non-targeted or targeted (e.g., oligonucleotide-directed) mutagenesis can be performed to introduce mutations into the TCR sequence, and the resulting TCRs can then be screened for affinity to the target, for example, by using phage display.

[0105] The terms "activate a T cell response with a signal-to-noise ratio that is stronger than or equal to a patient-derived MAGE-A4-specific TCR" or "activate a T cell response with a signal-to-noise ratio that is stronger than or equal to an affinity-matured MAGE-A4-specific TCR" are meant to refer to an increase, i.e., about 2-fold or more, an amplification, i.e., about 2-fold, an enhancement, i.e., about 2-fold, or a boost in physiological activity, i.e., about 2-fold, of T cell signaling, as measured, for example, by a luminescence bioassay. References to a greater T cell response or a stronger T cell response or activation signal may be used interchangeably. Various measurements and assays of T cell responses or T cell activation are well known to those of skill in the art.

[0106] By the phrase "therapeutically effective amount" is meant an amount that, when administered, produces a desired effect. The precise amount will depend on the purpose of the treatment and will be ascertainable by one of ordinary skill in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). The term "effective amount" is intended to encompass contexts such as a pharmaceutically effective amount or a therapeutically effective amount. For example, in certain embodiments, an effective amount can achieve a beneficial condition, a beneficial outcome, functional activity in a screening assay, or improvement in a clinical condition.

[0107] As used herein, the term "subject" refers to an animal, preferably a mammal, in need of amelioration, prevention, and / or treatment of a MAGE-A4-associated disease or disorder, such as a MAGE-A4-associated cancer (e.g., a MAGE-A4-positive cancer). The term includes human subjects having or at risk of having a MAGE-A4-associated disease or disorder, such as a MAGE-A4-associated cancer.

[0108] As used herein, "anticancer agent" refers to any agent that is useful for treating, ameliorating, or inhibiting cancer, and includes, but is not limited to, cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, cyclophosphamide, mitotane (O,P'-(DDD)), biologics (e.g., antibodies and interferons), and radioactive agents. As used herein, "cytotoxin or cytotoxic agent," which also refers to chemotherapeutic agents, refers to any agent that is harmful to cells. Examples include Taxol® (paclitaxel), temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinbiastine, coitisin, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof.

[0109] The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disorder or condition in a subject who does not have the disorder or condition but who is at risk of or susceptible to developing the disorder or condition. Prevention and the like does not mean preventing a subject from ever contracting a particular disease or disorder. Prevention may require the administration of multiple doses. Prevention can include preventing the recurrence of disease in a subject in which all disease symptoms have been eliminated, or preventing recurrence in relapsing-remitting disease.

[0110] II. MAGE-A4 T Cells (TCR) and Compositions Comprising MAGE-A4 TCR T cells are a subgroup of cells that, along with other immune cell types (polymorphonuclear, eosinophilic, basophilic, mast, B, and NK cells), constitute the cellular component of the immune system. Under physiological conditions, T cells function in immune surveillance and the elimination of foreign antigens. However, there is compelling evidence that under pathological conditions, T cells play a major role in the causation and propagation of disease. In these disorders, the disruption of either central or peripheral T cell tolerance is a fundamental process in the pathogenesis of autoimmune diseases.

[0111] T cells bind to epitopes at small antigenic determinants on the surface of antigen-presenting cells in association with major histocompatibility complex (MHC, mouse) or human leukocyte antigen (HLA, human) complexes. T cells bind to these epitopes through the T cell receptor (TCR) complex on the surface of the T cell. T cell receptors are heterodimeric structures consisting of two chains: α (alpha) and β (beta) chains, or γ (gamma) and δ (delta) chains. The α chain is encoded by a nucleic acid sequence located within the α locus (on human or mouse chromosome 14), which also encompasses the entire δ locus, and the β chain is encoded by a nucleic acid sequence located within the β locus (on mouse chromosome 6 or human chromosome 7). The majority of T cells possess αβ TCRs, while a minority possess γδ TCRs.

[0112] The T cell receptor α and β polypeptides (similarly, γ and δ polypeptides) are linked to each other via disulfide bonds. Each of the two polypeptides that make up the TCR contains an extracellular domain that includes constant and variable regions, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and cytoplasmic tail are also part of the constant region). The variable region of the TCR determines its antigen specificity and, like immunoglobulins, contains three complementarity-determining regions (CDRs). TCRs are expressed on most T cells in the body and are known to be involved in the recognition of MHC-restricted antigens. The TCR α chain contains covalently linked Vα and Cα regions, while the β chain contains a Vβ region covalently linked to the Cβ region. The Vα and Vβ regions form a pocket or cleft that can bind antigen in the context of the major histocompatibility complex (MHC) (or HLA in humans). TCRs are detection molecules with exquisite specificity and, like antibodies, exhibit high levels of diversity.

[0113] The general structure of TCR molecules, and methods of making and using them, including binding to peptide:major histocompatibility complexes, have been disclosed, see e.g., PCT / US98 / 04274, PCT / US98 / 20263, WO99 / 60120.

[0114] Non-human animals (e.g., rodents, e.g., mice or rats) can be genetically engineered to express human or humanized T cell receptors (TCRs) comprising a variable domain encoded by at least one human TCR variable region gene segment, as described, for example, in PCT Publication WO2016 / 164492, the entire contents of which are incorporated herein by reference. For example, the TCRs of the present invention can be produced using Veloci-T® mouse technology (Regeneron), a genetically engineered mouse that enables the production of fully human therapeutic TCRs against tumor and / or viral antigens. One skilled in the art can obtain mutated TCR sequences and test them for specific binding affinity and / or specificity through standard mutagenesis techniques in conjunction with the assays described herein. Useful mutagenesis techniques known in the art include, but are not limited to, de novo gene synthesis, oligonucleotide-directed mutagenesis, region-specific mutagenesis, linker scanning mutagenesis, and site-directed mutagenesis by PCR (see, for example, Sambrook et al. (1989) and Ausubel et al. (1999)).

[0115] Briefly, in one embodiment, a method for generating a TCR against a MAGE-A4 230-239 peptide or a MAGE-A4 286-294 peptide may comprise immunizing a non-human animal (e.g., a rodent, e.g., a mouse or rat), such as a genetically engineered non-human animal comprising an unrearranged human TCR variable locus in its genome, with a MAGE-A4 230-239 peptide or a MAGE-A4 286-294 peptide, mounting an immune response in the animal against the peptide, isolating T cells from the animal that react to the peptide, determining the nucleic acid sequence of the human TCR variable region expressed by the T cell, cloning the human TCR variable region into a nucleotide construct comprising the nucleic acid sequence of the human TCR constant region such that the human TCR variable region is operably linked to the human TCR constant region, and expressing from the construct a human T cell receptor specific for the MAGE-A4 230-239 peptide or the MAGE-A4 286-294 peptide, respectively. In one embodiment, the steps of isolating the T cells, determining the nucleic acid sequence of the human TCR variable region expressed by the T cells, cloning the human TCR variable region into a nucleotide construct comprising the nucleic acid sequence of the human TCR constant region, and expressing the human T cell receptor are performed using standard techniques known to those of skill in the art.

[0116] In one embodiment, a nucleotide sequence encoding a T cell receptor specific for an antigen of interest is expressed in a cell, hi one embodiment, the TCR-expressing cell is selected from CHO, COS, 293, HeLa, PERC.6™ cells, etc.

[0117] In obtaining a variant TCR coding sequence, those skilled in the art will recognize that TCR-derived proteins can be modified by specific amino acid substitutions, additions, deletions, and post-translational modifications without losing or reducing biological activity.In particular, it is well known that conservative amino acid substitutions, i.e., the substitution of one amino acid with another amino acid of similar size, charge, polarity, and conformation, are unlikely to significantly change protein function.The 20 standard amino acids that are the building blocks of proteins can be broadly divided into four groups of conserved amino acids: the nonpolar (hydrophobic) group includes alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine; the polar (uncharged, neutral) group includes asparagine, cysteine, glutamine, glycine, serine, threonine, and tyrosine; the positively charged (basic) group includes arginine, histidine, and lysine; and the negatively charged (acidic) group includes aspartic acid and glutamic acid. Substitution of one amino acid for another within the same group in a protein is unlikely to have an adverse effect on the biological activity of the protein.

[0118] In some embodiments, the TCRs of the present disclosure can comprise a CDR sequence (e.g., a CDR3 sequence such as a VαCDR3 or a VβCDR3) that has one or more substitutions compared to the CDR sequences in Table 6 (e.g., a CDR3 sequence such as a VαCDR3 or a VβCDR3). For example, the TCRs of the present disclosure can comprise a CDR sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions compared to the CDR sequences in Table 6. Generally, the TCRs of the present invention function by binding to the HLA-A2-presented MAGE-A4 230-239 peptide or the HLA-A2-presented MAGE-A4 286-294 peptide. As used herein, an HLA-presented peptide (e.g., an HLA-A2-presented peptide) can refer to a peptide that is bound to a human leukocyte antigen (HLA) protein, e.g., an HLA protein expressed on the surface of a cell. Thus, a TCR that binds to an HLA-presented peptide binds to a peptide bound by the HLA, and optionally also binds to the HLA itself. Interaction with the HLA can confer specificity for binding to a peptide presented by a particular HLA. In some embodiments, the TCR binds to an isolated HLA-presented peptide. In some embodiments, the TCR binds to an HLA peptide presented on the surface of a cell.

[0119] The present invention includes MAGE-A4 TCRs that bind with high specificity to MAGE-A4 230-239 peptide or MAGE-A4 286-294 peptide in the context of HLA-A2. In some embodiments, the MAGE-A4 TCR does not bind, or only minimally binds, to MAGE-A4 230-239 peptide or MAGE-A4 286-294 peptide in the absence of HLA-A2. Furthermore, in some embodiments, the MAGE-A4 TCR does not bind, or only minimally binds, to off-target peptides in the context of HLA-A2. As used herein, an off-target peptide can refer to a peptide that differs from the target peptide by one, two, three, four, five, or more amino acids. In some embodiments, binding specificity can be determined by a) measuring on-target binding (e.g., binding to an HLA-A2-presented MAGE-A4(230-239) peptide or an HLA-A2-presented MAGE-A4(230-239) peptide), b) measuring off-target binding, and c) quantifying the difference between the two, e.g., by calculating a ratio. This ratio can be calculated, e.g., by dividing the values ​​obtained in a) and b). Measuring on-target and off-target binding can be achieved, e.g., by measuring % binding to a peptide / HLA tetramer reagent (e.g., a MAGE-A4 / HLA tetramer reagent or a MAGE-A8 / HLA tetramer reagent) or by other techniques known in the art.In some embodiments, the on-target binding / off-target binding value (e.g., the value obtained by dividing the values ​​obtained in a) and b) above) of a TCR of the disclosure is greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, greater than 10, greater than 11, greater than 12, greater than 13, greater than 14, greater than 15, greater than 16, greater than 17, greater than 18, greater than 19, greater than 20, greater than 21, greater than 22, greater than 23, greater than 24, greater than 25, greater than 26, greater than 27, greater than 28, greater than 29, greater than 30, greater than 35, greater than 40, greater than 45, greater than 50, greater than 55, greater than 60, greater than 65, greater than 70, greater than 75, greater than 80, greater than 85, greater than 90, 95, greater than 100 greater than 110, greater than 120, greater than 130, greater than 140, greater than 150, greater than 160, greater than 170, greater than 180, greater than 190, greater than 200, greater than 225, greater than 250, greater than 275, greater than 300, greater than 325, greater than 350, greater than 375, greater than 400, greater than 425, greater than 450, greater than 475, greater than 500, greater than 550, greater than 600, greater than 650, greater than 700, greater than 750, greater than 800, greater than 850, greater than 900, greater than 950, greater than 1000, greater than 1100, greater than 1200, greater than 1300, greater than 1400, greater than 1500, greater than 1600, greater than 1700, greater than 1800, greater than 1900, or greater than 2000. In some embodiments, the on-target binding / off-target binding value (e.g., the value obtained by dividing the values ​​obtained in a) and b) above) can be about 5 to about 20, about 10 to about 30, about 20 to about 80, about 30 to about 70, about 40 to about 60, about 50 to about 250, about 100 to about 200, about 100 to about 1000, about 300 to about 700, about 500 to about 1500, about 800 to about 1200, about 900 to about 1100, about 800 to about 1500, about 1000 to about 1400, or about 1100 to about 1300.

[0120] In one embodiment, the present invention provides a recombinant antigen binding protein (e.g., an isolated antigen binding protein) that specifically binds to a conformational epitope of HLA-A2-presented human MAGE-A4(286-294) peptide or a conformational epitope of HLA-A2-presented human MAGE-A4(230-239) peptide, wherein the antigen binding protein (a) binds to a monomeric HLA-A2:MAGE-A4(286-294) peptide or a monomeric MAGE-A4(230-239) peptide with a binding dissociation equilibrium constant (K) of less than about 20 nM as measured by a surface plasmon resonance assay at 25°C. D ) and (b) to a monomeric HLA-A2:MAGE-A4(286-294) peptide or a monomeric MAGE-A4(230-239) peptide with a binding dissociation equilibrium constant (K) of less than about 25 nM as measured by a surface plasmon resonance assay at 25°C. D ) and (c) binding to HLA-A2:MAGE-A4(286-294) peptide-expressing cells or MAGE-A4(230-239) peptide-expressing cells with an EC of less than about 6 nM as determined by a luminescence assay. 50 and (d) bind to HLA-A2:MAGE-A4(286-294) peptide-expressing cells or MAGE-A4(230-239) peptide-expressing cells with an EC of less than about 1 nM, as determined by a luminescence assay. 50 and (e) binds to HLA-A2:MAGE-A4(286-294) peptide-expressing cells or MAGE-A4(230-239) peptide-expressing cells and has an EC of less than about 30 nM as determined by flow cytometry assay. 50 and (f) binding to HLA-A2:MAGE-A4(286-294) peptide-expressing cells or MAGE-A4(230-239) peptide-expressing cells with an EC of less than about 75 nM as determined by a flow cytometry assay. 50(g) mediating the killing of cancer cells (e.g., melanoma cells) in a dose-dependent manner as determined by a calcein AM dye release assay; and (h) the conformational epitope comprises one or more amino acids of SEQ ID NO: 611 or SEQ ID NO: 612.

[0121] In some embodiments, the MAGE-A4 TCR of the present disclosure has specific activity or affinity for MAGE-A4(230-239) or MAGE-A4(286-294) as measured by in vitro assays. For example, HLA-expressing cells (such as T2 cells) can be pulsed with MAGE-A4(230-239) or MAGE-A4(286-294) polypeptides, or off-target polypeptides, thereby inducing the cells to present polypeptides that bind to the HLA. Alternatively, or in addition to using off-target polypeptides as a control, an off-target HLA (an HLA other than the HLA recognized by the TCR of interest) can be used. For example, an off-target HLA can be used to present MAGE-A4 peptides and test the specificity of binding to HLA-A2-presented MAGE-A4 peptides. Additionally, the control can be a cell line that expresses neither MAGE-A4 nor the target HLA (e.g., HLA-A2). The cells can be co-cultured with a T cell population expressing the TCR of interest, and activity is measured as a function of the amount of cytokine (such as interferon-γ) produced by the cells. In certain embodiments, the assay is performed using an effector cell:target cell ratio of 1:1 (1 x 10 5 TCR-expressing T cell populations at 10 effector cells / 96 wells and 10 -10 In vitro co-culture of M with peptide-loaded T2 cells and interferon-γ measurement 24 hours after co-culture (e.g., Meso Scale Discovery (by MSD® Sector Imager). In certain embodiments, the assay is performed using an effector cell:target cell ratio of 5:1 (2.5×10 5Effector cells: 5 x 10 4 In vitro assays involving such cells can include in vitro co-culture of a TCR-expressing T cell population with effector cells on a 100-well plate (100-well plate) of target cells, and measurement of interferon-γ (e.g., by Meso Scale Discovery (MSD® Sector Imager)) 24 hours after co-culture. In certain embodiments, the TCR is expressed in primary T cells (e.g., cytotoxic T cells, regulatory T cells, helper T cells, or any combination thereof) by replacing an endogenous locus, e.g., an endogenous TRAC locus (KO / KI), and disrupting an endogenous locus, e.g., a TRBC locus (KO), using, e.g., CRISPR, TALEN, zinc finger, or other targeted disruption system. In vitro assays involving such cells can include cytotoxic activity against HLA-A2*01MAGE-A4-expressing A375 melanoma cells in a calcein AM dye release assay.

[0122] The increase in the amount of cytokine detected can be used as an indicator of activity. The activity or specificity of the TCR of interest for its target peptide compared to a control (off-target), or the activity or specificity of the TCR of interest for its on-target HLA-binding target peptide compared to an off-target HLA-binding target peptide can be 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 15-fold or more, 20-fold or more, 30-fold or more, 40-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 4-fold or more, 50-fold or more, 600-fold or more, 700-fold or more, 800-fold or more, 900-fold or more, 10 ... 00x or more, 500x or more, 600x or more, 700x or more, 800x or more, 900x or more, 1,000x or more, 1,500x or more, 2,000x or more, 2,500x or more, 3,000x or more, 4,000x or more, 5,000x or more, 10,000x or more, 20,000x or more, 30,000x or more, 40,000x or more, 50,000x or more, 60,000x or more, 70,000x or more, 80,000x or more, 90,000x or more, or 100,000x or more.

[0123] In certain embodiments, the MAGE-A4 TCRs of the present disclosure, when administered prophylactically to a subject in need thereof, are useful for inhibiting tumor growth or delaying the progression of cancer and may increase the survival of the subject. For example, administration of the MAGE-A4 TCRs of the present disclosure may result in the shrinkage of a primary tumor and may prevent the development of metastasis or secondary tumors. In certain embodiments, the MAGE-A4 TCRs of the present disclosure, when administered therapeutically to a subject in need thereof, are useful for inhibiting tumor growth and may increase the survival of the subject. For example, administration of a therapeutically effective amount of the MAGE-A4 TCRs of the present disclosure to a subject may result in the shrinkage and elimination of an established tumor in the subject.

[0124] In one embodiment, the invention provides a TCR (e.g., an isolated TCR, or a TCR expressed on an isolated cell) that specifically binds to an HLA-A2-presented MAGE-A4 286-294 peptide, wherein the antigen binding protein comprises (i) an alpha chain variable domain comprising complementarity determining regions (CDRs) 1, CDR2, and CDR3, wherein the CDR3 region is represented by the formula I: N1-N2-N3-N4-N5-N6-N7-N8-N9-N 10 -N 11 -N 12 -N 13 -N 14 -N 15N1 is Ala, Ile, or Gly; N2 is Val, but may be present or absent; N3 is Tyr, Gly, Leu, Val, Glu, Met, Ala, or Phe; N4 is Arg, Glu, Ser, Asn, Gln, Lys, Asp, Gly, or Met; and N5 is Ser, Arg, Glu, Leu, Ala, Asp, Pro, Met, Gly, or Lys, but may be present or absent. N6 is Ala, Asp, Gly, Ser, Val, Pro, Leu, Tyr, or Thr, but may be present or absent; N7 is Thr, Pro, Ser, Glu, Asp, Trp, Arg, Asn, Ile, Gln, or Leu, but may be present or absent; N8 is Gly, Trp, Thr, Lys, Tyr, or Ala; N9 is Asn, Gly, Lys, Ile, Ser, or Arg; 10 is Gln, Lys, Gly, Thr, Leu, Asp, or Ser, but may be present or absent, and N 11 is Phe, Asn, Thr, Tyr, Ala, Leu, Met, or Glu, but may be present or absent, and N 12 is Lys, Phe, Tyr, or Asp, but may be present or absent, and N 13 is Lys or Gly, but may be present or absent, and N 14 is Thr, Leu, or Tyr, but may be present or absent, and N 15 (ii) a beta chain variable domain comprising a beta chain variable domain comprising complementarity determining regions (CDR) 1, CDR2, and CDR3, wherein the CDR3 region is represented by the formula II: N1-N2-N3-N4-N5-N6-N7-N8-N9-N 10 -N 11 -N 12 -N 13 -N 14 -N 15 -N 16 -N 17 -N 18N1 is Ala or Ser, N2 is Ala, Ser, or Thr, N3 is Ser, Gly, or Trp, N4 is Leu, Tyr, Trp, Asp, Phe, Gly, Pro, or His, N5 is Gly or Asp, but may be present or absent, N6 is Phe or Arg, but may be present or absent, N7 is Trp, Phe, Asp, Pro, Tyr, Gly, Thr, Ser, or Val, but may be present or absent, N8 is Pro, Arg, Asp, Tyr, Gln, Asn, or Gly, but may be present or absent, N9 is Asp, but may be present or absent, and N 10 is Arg, but may be present or absent, and N 11 is Gly, Ala, or Thr, but may be present or absent, and N 12 is Ser, Trp, Thr, Gly, Val, Leu, Arg, Met, Tyr, or Gln, and N 13 is Gly, but may be present or absent, and N 14 is Asn, Asp, Gly, Thr, Pro, Gln, or His, but may be present or absent, and N 15 is Thr, Ser, Glu, Asn, Tyr, Gln, Asp, or Pro, but may be present or absent, and N 16 is Glu, Pro, Lys, Thr, Ala, Gly, or Gln, but may be present or absent, and N 17 is Ala, Leu, Ile, Tyr, or Gln, but may be present or absent, and N 18is Phe, His, Tyr, or Thr; (iii) a CDR1 of the alpha chain variable domain comprising any one of the CDR1 amino acid sequences shown in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and a CDR2 of the alpha chain variable domain independently comprising any one of the CDR2 amino acid sequences shown in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. (iv) a CDR1 of the beta chain variable domain comprising any one of the CDR1 amino acid sequences set forth in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and a CDR2 of the beta chain variable domain independently comprising any one of the CDR2 amino acid sequences set forth in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. (v) an alpha chain variable domain CDR1, CDR2, and CDR3 contained within any one of the alpha chain variable domain sequences listed in Table 4, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, with any one of the beta chain variable domain sequences listed in Table 4. (vi) a beta chain variable domain CDR1, CDR2, and CDR3 contained within any one of the alpha chain variable domain amino acid sequences listed in Table 4, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (vi) a beta chain variable domain CDR1, CDR2, and CDR3 contained within any one of the alpha chain variable domain amino acid sequences listed in Table 4, or a substantially similar sequence thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,(vii) a beta chain variable domain having an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta chain variable domain amino acid sequences listed in Table 4; (viii)(a) an alpha chain variable domain having an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta chain variable domain amino acid sequences listed in Table 4; and (b) an alpha chain variable domain having an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the main amino acid sequences; and (c) an alpha chain variable domain having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta chain variable domain amino acid sequences listed in Table 4. and (ix) (a) a beta chain variable domain having an amino acid sequence with 5%, 96%, 97%, 98%, 99%, or about 100% amino acid identity to a target polypeptide; and (b) a beta chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337, 353, 369, 385, 401, 417, 433, 449, 465, 481, 497, 513, 529, 545, 561, 577, and 593. (b) a CDR1 domain of a human F chain variable domain, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and (c) a human F chain variable domain CDR1 domain of a human F chain variable domain, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and (d) a human F chain variable domain CDR1 domain of a human F chain variable domain, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, andand 594, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and (c) SEQ ID NOs: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307, 323, 339, 355, 371, 387, 403, 419, 435, 451, 467. , 483, 499, 515, 531, 547, 563, 579, and 595, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and (d) an alpha chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, 329, 345, 36 (e) a beta chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 377, 393, 409, 425, 441, 457, 473, 489, 505, 521, 537, 553, 569, 585, and 601, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and (f) a beta chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 262, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 372, 380, 392, 400, 425, 441, 457, 473, 489, 505, 521, 537, 553, 569, 585, and 601, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. (f) a beta chain variable domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 160, 161, 162, 163, 164, 165, 166, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554, 570, 586, and 602, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;171、187、203、219、235、251、267、283、299、31、 (x) a beta chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 / 15, 23 / 31, 39 / 47, 55 / 63, 71 / 79, 87 / 95, 103 / 104, 115 / 116, 125 / 128, 135 / 139, 140 / 142, 145 / 146, 150 / 152, 155 / 153, 165 / 166, 167 / 168, 170 / 172, 175 / 176, 177 / 178, 180 / 184, 185 / 186, 187 / 188, 189 / 190, 191 / 192, 193 / 194, 194 / 196, 195 / 196, 200 / 200, 201 / 202, 202 / 203, 203 / 204, 205 / 206, 206 / 208, 210 / 212, 211 / 214, 212 / 216, 213 / 218, 214 / 219, 220 / 222, 225 / 226, 226 / 228, 230 / 230, 231 / 347, 363, 379, 395, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555, 571, 587, and 603, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 11, 119 / 127, 135 / 143, 151 / 159, 167 / 175, 183 / 191, 199 / 207, 215 / 223, 231 / 239, 247 / 255, 263 / 271, 279 / 287, 295 / 303, 311 / 319, 327 / 335, 343 / 351, 35 9 / 367, 375 / 383, 391 / 399, 407 / 415, 423 / 431, 439 / 447, 455 / 463, 471 / 479, 487 / 495, 503 / 511, 519 / 527, 535 / 543, 551 / 559, 567 / 575, 583 / 591, 599 / 607 (xi) an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of SEQ ID NOs: 87 / 31, 23 / 95, 231 / 607, 231 / 223, 231 / 591, 231 / 255, 231 / 271, 231 / 79, 231 / 47, 231 / 399, 599 / 239, 599 / 223, 599 / 591, 599 / 255, 599 / 27 1, 599 / 79, 599 / 47, 599 / 399, 215 / 239, 215 / 607, 215 / 591, 215 / 255, 215 / 271, 215 / 79, 215 / 47, 215 / 399, 583 / 239, 583 / 607, 583 / 223, 583 / 255, 583 / 271 , 583 / 79, 583 / 47, 583 / 399, 247 / 239, 247 / 607, 247 / 223, 247 / 591, 247 / 271, 247 / 79, 247 / 47, 247 / 399, 263 / 239, 263 / 607, 263 / 223, 263 / 591, 263 / 255,263 / 79、263 / 47、263 / 399、71 / 239、71 / 607、71 / 223、71 / 591、71 / 255、71 / 271、71 / 47、71 / 399、39 / 239、39 / 607、39 / 223、39 / 591、39 / 255、39 / 271、39 / 79、39 / 399、391 / 239、391 / 607、391 / 223、391 / 591、391 / 255、391 / 271、391 / 79、391 / 47、439 / 127、439 / 319、439 / 287、439 / 15、439 / 111、439 / 383、439 / 191、439 / 511、439 / 527、439 / 559、439 / 207、119 / 447、119 / 319、119 / 287、119 / 15、119 / 111、119 / 383、119 / 191、119 / 511、119 / 527、119 / 559、119 / 207、311 / 447、311 / 127、311 / 287、311 / 15、311 / 111、311 / 383、311 / 191、311 / 511、311 / 527、311 / 559、311 / 207、279 / 447、279 / 127、279 / 319、279 / 15、279 / 111、279 / 383、279 / 191、279 / 511、279 / 527、279 / 559、279 / 207、7 / 447、7 / 127、7 / 319、7 / 287、7 / 111、7 / 383、7 / 191、7 / 511、7 / 527、7 / 559、7 / 207、103 / 447、103 / 127、103 / 319、103 / 287、103 / 15、103 / 383、103 / 191、103 / 511、103 / 527、103 / 559、103 / 207、375 / 447、375 / 127、375 / 319、375 / 287、375 / 15、375 / 111、375 / 191、375 / 511、375 / 527、375 / 559、375 / 207、183 / 447、183 / 127、183 / 319、183 / 287、183 / 15、183 / 111、183 / 383、183 / 511、183 / 527、183 / 559、183 / 207、503 / 447、503 / 127、503 / 319、503 / 287、503 / 15、503 / 111、503 / 383、503 / 191、503 / 527、503 / 559、503 / 207、519 / 447、519 / 127、519 / 319、519 / 287、519 / 15、519 / 111, 519 / 383, 519 / 191, 519 / 511, 510 / 559, 519 / 207, 551 / 447, 551 / 127, 551 / 319, 551 / 287, 551 / 15, 551 / 111, 551 / 383, 551 / 191, 551 / 511, 551 / 527, 551 / 207, 199 / 447, 199 / 127, 199 / 319, 199 / 287, 199 / 15, 199 / 111, 199 / 383, 199 / 191, 199 / 511 , 199 / 527, and 199 / 559, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and / or (xii) the lack of binding to cells expressing predicted off-target peptides.

[0125] In one embodiment, the present invention provides HLA-A2-presenting MAGE-A4 and a CDR2 of the alpha chain variable domain independently comprising any one of the CDR2 amino acid sequences set forth in Table 6, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and (iv) a CDR1 of the beta chain variable domain comprising any one of the CDR1 amino acid sequences set forth in Table 6, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. and a substantially similar sequence thereof with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (v) an alpha chain variable domain CDR1, CDR2, and CDR3 comprised within any one of the alpha chain variable domain sequences listed in Table 8, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and a beta chain variable domain CDR1, CDR2, and CDR3 comprised within any one of the beta chain variable domain sequences listed in Table 8, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;(vi) comprising an alpha chain variable domain having an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 8; (vii) at least 85%, 86%, 87%, 88%, to the entire amino acid sequence of any one of the amino acid sequences of the beta chain variable domain amino acid sequences listed in Table 8; (viii) (a) a beta chain variable domain having an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 8. and (b) a beta chain variable domain having an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% amino acid identity to the entire amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 8; (ix) a beta chain variable domain having (a) a beta chain variable domain selected from the group consisting of SEQ ID NOs: 614, 630, 646, 662, 678, 694, 710, 726, 742, 758, 774, 790, 806, 822, 832, 842, 852, 862, 872, 882, 892, 904, 910, 926, 932, 942, 952, 962, 972, 982, 9 ... (b) an alpha chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 615, 631, 647, 663, 679, 695, 711, 727, 743, 759, 775, 791, 807, 823, 839, and 855, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; andor a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and (c) an alpha chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 616, 632, 648, 664, 680, 696, 712, 728, 744, 760, 776, 792, 808, 824, 840, and 856, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. (d) a beta chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 622, 638, 654, 670, 686, 702, 718, 734, 750, 766, 782, 798, 814, 830, 846, and 862, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and (e) a beta chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 623, 639, 655, 671, 686, 702, 718, 734, 750, 766, 782, 798, 814, 830, 846, and 862, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. , 687, 703, 719, 735, 751, 767, 783, 799, 815, 831, 847, and 863, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and (f) a beta chain variable domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 624, 640, 656, 672, 688, 704, 720, 736, 752, 768, 784, 800, 816, 832, 848, and 864. and (x) a beta chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 620 / 628, 636 / 644, 652 / 660, 668 / 676, 684 / 692, 700 / 708, 716 / 724, 732 / 740, 748 / 756, 764 / 772, 780 / 788, 796 / 804, 812 / 820, 828 / 836, 844 / 852, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;and 860 / 868, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (xi) SEQ ID NOs: 620 / 628, 620 / 644, 620 / 660, 620 / 676, 620 / 692, 620 / 708, 620 / 724, 620 / 740, 620 / 756, 620 / 772, 620 / 788, 620 / 804, 620 / 82 0, 620 / 836, 620 / 852, 620 / 868, 636 / 628, 636 / 644, 636 / 660, 636 / 676, 636 / 692, 636 / 708, 636 / 724, 636 / 740, 636 / 756, 636 / 772, 636 / 788, 636 / 804, 63 6 / 820, 636 / 836, 636 / 852, 636 / 868, 652 / 628, 652 / 644, 652 / 660, 652 / 676, 652 / 692, 652 / 708, 652 / 724, 652 / 740, 652 / 756, 652 / 772, 652 / 788, 652 / 804 , 652 / 820, 652 / 836, 652 / 852, 652 / 868, 668 / 628, 668 / 644, 668 / 660, 668 / 676, 668 / 692, 668 / 708, 668 / 724, 668 / 740, 668 / 756, 668 / 772, 668 / 788, 668 / 804, 668 / 820, 668 / 836, 668 / 852, 668 / 868, 684 / 628, 684 / 644, 684 / 660, 684 / 676, 684 / 692, 684 / 708, 684 / 724, 684 / 740, 684 / 756, 684 / 772, 684 / 788, 684 / 804, 684 / 820, 684 / 836, 684 / 852, 684 / 868, 700 / 628, 700 / 644, 700 / 660, 700 / 676, 700 / 692, 700 / 708, 700 / 724, 700 / 740, 700 / 756, 700 / 772, 700 / 788, 700 / 804, 700 / 820, 700 / 836, 700 / 852, 700 / 868, 716 / 628, 716 / 644, 716 / 660, 716 / / 676, 716 / 692, 716 / 708, 716 / 724, 716 / 740, 716 / 756, 716 / 772,716 / 788、716 / 804、716 / 820、716 / 836、716 / 852、716 / 868、732 / 628、732 / 644、732 / 660、732 / 676、732 / 692、732 / 708、732 / 724、732 / 740、732 / 756、732 / 772、732 / 788、732 / 804、732 / 820、732 / 836、732 / 852、732 / 868、748 / 628、748 / 644、748 / 660、748 / 676、748 / 692、748 / 708、748 / 724、748 / 740、748 / 756、748 / 772、748 / 788、748 / 804、748 / 820、748 / 836、748 / 852、748 / 868、764 / 628、764 / 644、764 / 660、764 / 676、764 / 692、764 / 708、764 / 724、764 / 740、764 / 756、764 / 772、764 / 788、764 / 804、764 / 820、764 / 836、764 / 852、764 / 868、780 / 628、780 / 644、780 / 660、780 / 676、780 / 692、780 / 708、780 / 724、780 / 740、780 / 756、780 / 772、780 / 788、780 / 804、780 / 820、780 / 836、780 / 852、780 / 868、796 / 628、796 / 644、796 / 660、796 / 676、796 / 692、796 / 708、796 / 724、796 / 740、796 / 756、796 / 772、796 / 788、796 / 804、796 / 820、796 / 836、796 / 852、796 / 868、812 / 628、812 / 644、812 / 660、812 / 676、812 / 692、812 / 708、812 / 724、812 / 740、812 / 756、812 / 772、812 / 788、812 / 804、812 / 820、812 / 836、812 / 852、812 / 868、828 / 628、828 / 644、828 / 660、828 / 676、828 / 692、828 / 708、828 / 724、828 / 740、828 / 756、828 / 772、828 / 788、828 / 804、828 / 820、828 / 836、828 / 852、828 / 868、844 / 628、844 / 644、844 / 660、844 / 676、844 / 692、844 / 708、844 / 724、844 / 740, 844 / 756, 844 / 772, 844 / 788, 844 / 804, 844 / 820, 844 / 836, 844 / 852, 844 / 868, 860 / 628, 860 / 644, 860 / 660, 860 / 676, 860 / 692, 860 / 708, 860 / 724, 860 / 740, 860 / 756, 860 / 772, 860 / 788, 860 / 804, 860 / 820, 860 / , and / or (xii) exhibiting one or more of the following characteristics: (i) comprising an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of 836, 860 / 852, and 860 / 868, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and / or (xiii) not binding to cells expressing predicted off-target peptides.

[0126] The TCRs of the present invention may have one or more of the above biological characteristics, or any combination thereof. Other biological characteristics of the antigen-binding proteins of the present invention will be apparent to those skilled in the art from a review of the present disclosure, including the working examples herein.

[0127] In certain embodiments, the polynucleotide encoding the MAGE-A4 TCR described herein is inserted into a vector. As used herein, the term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be covalently inserted to result in expression of the protein and / or cloning of the polynucleotide. Such vectors may also be referred to as "expression vectors." An isolated polynucleotide can be inserted into a vector using any suitable method known in the art, for example, but not limited to, a vector can be digested with an appropriate restriction enzyme and then ligated to an isolated polynucleotide with matching restriction ends. Expression vectors are capable of incorporating and expressing heterologous or modified nucleic acid sequences that encode at least a portion of a gene product that can be transcribed intracellularly. In most cases, the RNA molecule is then translated into a protein. Expression vectors can contain various control sequences, which refer to nucleic acid sequences necessary for the transcription or translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences that perform other functions as well, which are discussed below. An expression vector may contain additional elements, for example, it may have two replication systems, thus allowing it to be maintained in two organisms, for example, human cells for expression and a prokaryotic host for cloning and amplification.

[0128] For efficient gene transcription and translation in their respective host cells, expression vectors may contain necessary 5' upstream and 3' downstream regulatory elements, such as promoter sequences such as CMV, PGK, and EF1α promoters, ribosome recognition and binding TATA boxes, and 3' UTR AAUAAA transcription termination sequences. Other suitable promoters include the constitutive promoters of the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukosis virus promoter, EBV immediate early promoter, and Rous sarcoma virus promoter. Human gene promoters can also be used, including, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In certain embodiments, inducible promoters are also contemplated as part of vectors expressing chimeric antigen receptors. This provides a molecular switch that can turn on or off expression of the polynucleotide sequence of interest. Examples of inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, or a tetracycline promoter.

[0129] The expression vector may have additional sequences such as 6x histidine (SEQ ID NO: 871), c-Myc, and FLAG tags that are incorporated into the expressed TCR. Thus, the expression vector may be engineered to contain 5' and 3' untranslated regulatory sequences, which can sometimes function as enhancer sequences, promoter regions, and / or terminator sequences that can promote or enhance the efficient transcription of the nucleic acid of interest carried in the expression vector. The expression vector may also be engineered for replication and / or expression functionality (e.g., transcription and translation) in a specific cell type, cell location, or tissue type. The expression vector may contain a selectable marker for maintaining the vector in a host or recipient cell.

[0130] Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements.Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.Examples of animal virus categories that are useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors are the Lenti-X™ Bicistronic Expression System (Neo) vector (Clontrch) for expression in mammalian cells, pClneo vector (Promega), and pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequences of the TCRs disclosed herein can be ligated into such expression vectors for expression of chimeric proteins in mammalian cells.

[0131] In a specific embodiment, the nucleic acid encoding the TCR of the present invention is provided in a viral vector. The viral vector can be derived from a retrovirus, lentivirus, or foamy virus. As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. The viral vector can contain coding sequences for various proteins described herein in place of non-essential viral genes. The vector and / or particle can be utilized for the purpose of transferring DNA, RNA, or other nucleic acids into cells either in vitro or in vivo. Many forms of viral vectors are known in the art.

[0132] In certain embodiments, the viral vector that comprises the coding sequence of TCR described herein is retroviral vector or lentiviral vector.The term " retroviral vector " refers to the vector that comprises the structural and functional gene elements that are mainly derived from retrovirus.The term " lentiviral vector " refers to the vector that comprises the structural and functional gene elements outside LTR that are mainly derived from lentivirus.

[0133] Retroviral vectors for use herein can be derived from any known retrovirus (e.g., c-type retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV)). "Retrovirus" of the present invention also includes lentiviruses of the Retroviridae family, such as human T-cell leukemia viruses, HTLV-1 and HTLV-2, and human immunodeficiency viruses, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV), and other classes of retroviruses.

[0134] As used herein, lentiviral vector refers to a vector derived from a lentivirus, a group (or genus) of retroviruses that cause slowly developing diseases. Viruses included in this group include HIV (human immunodeficiency virus, including HIV types 1 and 2), visna-maedi, caprine arthritis-encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). Preparation of recombinant lentiviruses can be achieved using the methods described by Dull et al. and Zufferey et al. (Dull et al., J. Virol., 1998;72:8463-8471 and Zufferey et al., J. Virol., 1998;72:9873-9880).

[0135] Retroviral vectors (i.e., both lentiviral and non-lentiviral) for use in the present invention can be generated using standard cloning techniques by combining the desired DNA sequences with the instructions and adaptations described herein (Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals; Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145, Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043, Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381, Chowdhury et al. (1991) Science 254:1802-1805, van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644, Kay et al. (1992) Human Gene Therapy 3:641-647, Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895, Hwu et. al. (1993) J. Immunol 150:4104-4115, U.S. Patent No. 4,868,116, U.S. Patent No. 4,980,286, PCT Application WO89 / 07136, PCT Application WO89 / 02468, PCT Application WO89 / 05345, and PCT Application WO92 / 07573).

[0136] Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) sequences for use in generating vectors include genomic RNA and cDNA available from commercial sources including, for example, Type Culture Collection (ATCC), Rockville, Md. Sequences can also be chemically synthesized.

[0137] For expression of MAGE-A4 TCR, a vector can be introduced into a host cell to allow expression of the polypeptide within the host cell. Expression vectors can contain various elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements can be appropriately selected by those skilled in the art, as described above. For example, a promoter sequence can be selected to promote transcription of a polynucleotide in the vector. Suitable promoter sequences include, but are not limited to, T7 promoter, T3 promoter, SP6 promoter, β-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. An enhancer sequence can be selected to enhance transcription of a polynucleotide. A selectable marker can allow host cells into which a vector has been inserted to be selected from those without the vector; for example, a selectable marker can be a gene that confers antibiotic resistance. A signal sequence can be selected to allow the expressed polypeptide to be transported outside the host cell.

[0138] In cloning a polynucleotide, a vector is introduced into a host cell (isolated host cell) to allow the vector to replicate itself, thereby amplifying copies of the polynucleotide contained therein. Cloning vectors generally include, but are not limited to, a replication origin, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements can be appropriately selected by those skilled in the art. For example, a replication origin can be selected to promote the autonomous replication of the vector in the host cell.

[0139] In certain embodiments, the present disclosure provides an isolated host cell comprising a vector provided herein. Host cells comprising a vector may be useful for expressing or cloning a polynucleotide contained in the vector. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells. Suitable prokaryotic cells for this purpose include, but are not limited to, Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, including E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, including Salmonella typhimurium, Serratia, including Serratia marcescans, and Shigella, as well as Bacilli, including B. subtilis and B. licheniformis, Pseudomonas, including P. aeruginosa, and Streptomyces.

[0140] The TCR of the present invention is introduced into host cells using transfection and / or transduction techniques known in the art. As used herein, the terms "transfection" and "transduction" refer to the process by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid can be integrated into the host cell DNA or maintained extrachromosomally. The nucleic acid can be transiently maintained or stably introduced. Transfection can be achieved by various means known in the art, including, but not limited to, calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and gene guns. Transduction refers to the delivery of genes using viral or retroviral vectors by means of viral infection rather than transfection. In certain embodiments, retroviral vectors are transduced by packaging the vector into virions before contacting the cell. For example, a nucleic acid encoding a MAGE-A4 TCR of the present invention carried by a retroviral vector can be transduced into cells through infection and proviral integration.

[0141] As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms "genetically modified cell," "modified cell," and "reintroduced cell" are used interchangeably.

[0142] In particular, the TCRs of the present invention are introduced into and expressed in immune effector cells, thereby redirecting their specificity to a target antigen of interest, e.g., an HLA-A2-presented MAGE-A4 peptide, e.g., amino acid residues 230-239 or 286-294 of MAGE-A4.

[0143] The present invention provides methods for generating immune effector cells that express a TCR described herein. In one embodiment, the method comprises transfecting or transducing immune effector cells, e.g., immune effector cells isolated from a subject, such as a subject with a MAGE-A4-associated disease or disorder, whereby the immune effector cells express one or more TCRs described herein. In certain embodiments, immune effector cells are isolated from an individual and genetically modified without further in vitro manipulation. Such cells can then be directly readministered to the individual. In a further embodiment, immune effector cells are first activated and stimulated to proliferate in vitro, and then genetically modified to express a TCR. In this regard, the immune effector cells can be cultured before or after being genetically modified (i.e., transduced or transfected to express a TCR described herein).

[0144] Prior to in vitro manipulation or genetic modification of the immune effector cells described herein, a source of cells can be obtained from a subject. In particular, immune effector cells for use in the TCRs described herein include T cells.

[0145] T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic effusion, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction, and the cells can be placed in an appropriate buffer or medium for subsequent processing. In one embodiment, the cells are washed with PBS. In an alternative embodiment, the wash solution can lack calcium, magnesium, or many, but not all, divalent cations. As will be appreciated by those skilled in the art, the washing step can be accomplished by methods known to those skilled in the art, such as using a semi-automated flow-through centrifuge. After washing, the cells can be resuspended in a variety of biocompatible buffers or other saline solutions, with or without buffer. In certain embodiments, undesirable components of the apheresis sample can be removed directly in the medium in which the cells are resuspended.

[0146] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes, e.g., by centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of T cell populations by negative selection can be achieved by combining antibodies against surface markers unique to the negatively selected cells. One method for use herein is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry, which uses a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present invention.

[0147] PBMCs can be used directly for TCR genetic modification using the methods described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion.

[0148] Immune effector cells such as T cells can be genetically modified after isolation using known methods, or immune effector cells can be activated and expanded (or differentiated in the case of precursor cells) in vitro before being genetically modified.In another embodiment, immune effector cells such as T cells are genetically modified with the chimeric antigen receptor described herein (e.g., transduced with a viral vector containing a nucleic acid encoding a TCR), and then activated and expanded in vitro.Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Patent No. 6,905,874, U.S. Patent No. 6,867,041, U.S. Patent No. 6,797,514, WO2012 / 079000, and U.S. Patent Application Publication No. 2016 / 0175358.

[0149] The present invention provides a population of engineered immune effector cells for the treatment of a MAGE-A4 associated disease or disorder, such as cancer, wherein the engineered immune effector cells comprise a MAGE-A4 TCR as disclosed herein.

[0150] TCR-expressing immune effector cells prepared as described herein can be utilized in methods and compositions for adoptive immunotherapy according to known techniques or variations thereof that will be apparent to those skilled in the art based on this disclosure. See, e.g., U.S. Patent Application Publication No. 2003 / 0170238 by Gruenberg et al.; see also U.S. Patent No. 4,690,915 by Rosenberg.

[0151] III. Pharmaceutical Compositions The present invention provides therapeutic compositions comprising a MAGE-A4 TCR of the invention, or immune effector cells comprising a MAGE-A4 TCR of the invention. Therapeutic compositions according to the invention are administered with suitable carriers, excipients, and other agents that may be incorporated into the formulation to provide improved transport, delivery, tolerance, etc. Many suitable formulations are available from Remington's Pharmaceuticals, a formulary known to all pharmaceutical chemists. Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and carbowax-containing semi-solid mixtures. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0152] The frequency and duration of treatment can be adjusted depending on the severity of the condition.

[0153] In certain embodiments, the initial dose may be followed by administration of a second or multiple subsequent doses of the MAGE-A4 TCR of the present invention or immune effector cells comprising the MAGE-A4 TCR of the present invention in an amount approximately equal to or less than the initial dose.

[0154] In certain situations, the pharmaceutical composition can be delivered in a sustained release system, hi one embodiment, a pump can be used.

[0155] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal, and intramuscular injections, infusions, and the like. The TCRs, pharmaceutical compositions, and cells described herein can be administered parenterally. The preparations of the present disclosure can be prepared by publicly known methods. For example, the preparations can be prepared by dissolving, suspending, or emulsifying the antigen-binding protein or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, saline, glucose-containing isotonic solutions, and other adjuvants, which can be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media include, for example, sesame oil and soybean oil, which can be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection solution thus prepared is preferably filled into a suitable ampule.

[0156] In some embodiments, TCR-expressing immune effector cells are formulated by first harvesting them from their culture medium, then washing and concentrating the cells in a medium and container system (a "pharmaceutically acceptable" carrier) suitable for administration in a therapeutically effective amount. A suitable infusion medium can be any isotonic medium formulation, typically saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), although 5% dextrose in water or lactated Ringer's solution can also be utilized. The infusion medium can be supplemented with human serum albumin.

[0157] The therapeutically effective number of cells in the composition is typically greater than 10 cells 2 More than 10 6 10 cells 8 pieces or 10 9 Contains 10 cells 10The number of cells will depend on the end use for which the composition is intended and the cell types contained therein.

[0158] The cells can be autologous or heterologous to the patient receiving therapy. If desired, the treatment can also include administration of a mitogen (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18, and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) described herein to enhance the induction of an immune response.

[0159] The TCR-expressing immune effector cell populations of the present invention can be administered alone or as pharmaceutical compositions in combination with other components, such as diluents and / or IL-2 or other cytokines or cell populations. Briefly, pharmaceutical compositions of the present invention can comprise a TCR-expressing immune effector cell population, such as a T cell described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. Compositions of the present invention are preferably formulated for intravenous administration.

[0160] IV. THERAPEUTIC USES OF MAGE-A4 TCR OR IMMUNE EFFECTOR CELLS CONTAINING MAGE-A4 TCR The anti-tumor immune response induced in a subject by administering the TCR-expressing T cells described herein using the methods described herein or other methods known in the art can include cellular immune responses mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses, which can kill infected cells. A humoral immune response, primarily mediated by helper T cells, which can activate B cells and thus lead to antibody production, can also be induced. Various techniques can be used to analyze the type of immune response induced by the compositions of the present invention, and they are well described in the art, for example, Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0161] As such, the MAGE-A4 TCRs of the present invention are useful, inter alia, for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by MAGE-A4. For example, the present invention provides methods for treating (tumor growth inhibition) a MAGE-A4 associated disease or disorder, such as a MAGE-A4 associated cancer (e.g., a MAGE-A4 positive cancer), by administering to a patient in need of such treatment a MAGE-A4 TCR (or a pharmaceutical composition comprising a MAGE-A4 TCR or a plurality of cells comprising a MAGE-A4 TCR) as described herein, and a MAGE-A4 TCR (or a pharmaceutical composition comprising a MAGE-A4 TCR) for use in treating a MAGE-A4 associated cancer. The antigen binding proteins of the present invention are useful for the treatment, prevention, and / or amelioration of a disease or disorder or condition, such as a MAGE-A4 associated cancer, and / or for ameliorating at least one symptom associated with such a disease, disorder, or condition. In the context of the methods of treatment described herein, MAGE-A4 The TCR (or pharmaceutical composition or multiple cells) may be administered as a monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0162] Accordingly, the present invention provides a method for treating an individual diagnosed with, suspected of having, or at risk of developing a MAGE-A4 associated disease or disorder, such as a MAGE-A4 associated cancer, which method comprises administering to the individual a therapeutically effective amount of a TCR-expressing immune effector cell as described herein.

[0163] In one embodiment, the present invention provides a method of treating a subject diagnosed with a MAGE-A4 positive cancer, the method comprising removing immune effector cells from a subject diagnosed with a MAGE-A4 positive cancer, genetically modifying the immune effector cells with a vector comprising a nucleic acid encoding a TCR of the present invention, thereby producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject. In one embodiment, the immune effector cells comprise T cells.

[0164] Methods of administering the cell compositions described herein include any method effective to result in the reintroduction of ex vivo genetically modified immune effector cells, either directly expressing a TCR of the invention in the subject, or expressing a TCR upon the reintroduction of genetically modified precursor cells of immune effector cells that differentiate into mature immune effector cells upon introduction into the subject. One method involves transducing peripheral blood T cells ex vivo with a nucleic acid construct according to the invention and returning the transduced cells to the subject.

[0165] In some embodiments of the present invention, the compositions described herein are useful for treating a subject suffering from primary or recurrent cancer, including, but not limited to, MAGE-A4 associated cancers, for example, MAGE-A4 associated cancers include liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, metastatic solid tumor, colon cancer, stomach cancer, gastric cancer, In one embodiment, the MAGE-A4 associated cancer is ovarian cancer, melanoma, non-small cell lung cancer, hepatocellular carcinoma, colorectal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, stomach cancer, bladder cancer, head and neck cancer, gastric cancer, synovial sarcoma, or myxoid round cell liposarcoma.

[0166] The TCRs can be used to treat early or late-stage symptoms of MAGE-A4-associated cancer. In one embodiment, the TCRs of the present invention can be used to treat advanced or metastatic cancer. The TCRs are useful for reducing, inhibiting, or shrinking tumor growth. In certain embodiments, treatment with the TCRs of the present invention results in greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% regression of tumors in a subject. In certain embodiments, the TCRs can be used to prevent tumor recurrence. In certain embodiments, the TCRs are useful for extending progression-free survival or overall survival in subjects with MAGE-A4-associated cancer. In some embodiments, the TCRs are useful for reducing toxicity resulting from chemotherapy or radiation therapy while maintaining long-term survival in patients with MAGE-A4-associated cancer.

[0167] One or more TCRs of the invention may be administered to alleviate or prevent or reduce the severity of one or more symptoms or conditions of a disease or disorder.

[0168] The prophylactic use of one or more TCRs of the present invention in patients at risk of developing a disease or disorder, such as a MAGE-A4 associated disease or disorder, such as a MAGE-A4 associated cancer, is also contemplated herein.

[0169] In a further embodiment of the invention, the TCR is used for the preparation of a pharmaceutical composition for treating a patient suffering from a MAGE-A4 associated disease or disorder, such as a MAGE-A4 associated cancer. In another embodiment of the invention, the TCR is used as an adjunct therapy to any other agent or therapy known to those skilled in the art to be useful for the treatment of a MAGE-A4 associated cancer.

[0170] The combination therapy may comprise a MAGE-A4 TCR of the invention, such as an immune effector cell comprising a TCR of the invention, or a pharmaceutical composition of the invention, and any additional therapeutic agent that may be advantageously combined with the TCR of the invention. The TCRs of the present invention may be synergistically combined with one or more anti-cancer agents or therapies used to treat or inhibit a MAGE-A4 associated disease or disorder such as a MAGE-A4 positive cancer, for example liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, metastatic solid tumor, colon cancer, stomach cancer, gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, or recurrent non-small cell lung cancer.

[0171] It is contemplated herein that the TCRs of the present invention can be used in combination with immunostimulatory and / or immunosupportive therapy to inhibit tumor growth and / or improve the survival of cancer patients. Immunostimulatory therapy includes direct immunostimulatory therapy, which enhances immune cell activity by either "releasing the brakes" or "stepping on the gas" on suppressed immune cells to activate the immune response. Examples include targeting other checkpoint receptors, vaccination, and adjuvants. Immune supportive modalities may increase tumor antigenicity by promoting immunogenic cell death, inflammation, or have other indirect effects that promote anti-tumor immune responses. Examples include radiation, chemotherapy, antiangiogenic agents, and surgery.

[0172] In various embodiments, one or more TCRs of the invention are administered with a PD-1 inhibitor (e.g., an anti-PD-1 antibody such as nivolumab, pembrolizumab, pidilizumab, BGB-A317, or REGN2810), a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody such as avelumab, atezolizumab, durvalumab, MDX-1105, or REGN3504), a CTLA-4 inhibitor (e.g., ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T cell co-inhibitor or ligand (e.g., an antibody against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), an indoleamine-2,3,dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist [e.g., see U.S. Pat. No. 7,087,139,411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab, or ranibizumab), or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib)], Ang2 inhibitors (e.g., nesbacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens [e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9], vaccines (e.g., These include sterile Mycobacterium bovis, cancer vaccines, adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g., CD3xCD20 bispecific antibody or PSMAxCD3 bispecific antibody), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, surgery, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21, and IL-15, antibody-drug conjugates (e.g., anti-CD19-DM4 ADCs, and anti-DS6-DM4 ADCs), anti-inflammatory agents (e.g., corticosteroids, nonsteroidal anti-inflammatory agents), nutritional supplements such as antioxidants, or any other therapy for treating cancer. In certain embodiments, the TCRs of the present invention may be used in combination with cancer vaccines, including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, etc., to enhance anti-tumor responses.

[0173] Examples of cancer vaccines that can be used in combination with the TCRs of the present invention include MAGE3 vaccines for melanoma and bladder cancer, MUC1 vaccines for breast cancer, EGFRv3 (e.g., rindopepimut) for brain tumors (including glioblastoma multiforme), or ALVAC-CEA (for CEA+ cancers).

[0174] In certain embodiments, the MAGE-A4 TCRs of the present invention may be administered in combination with radiation therapy in a manner that produces a long-lasting anti-tumor response and / or enhances survival of patients with cancer. In some embodiments, the MAGE-A4 TCRs of the present invention may be administered before, simultaneously with, or after radiation therapy to a cancer patient. For example, radiation therapy may be administered in one or more doses to a tumor lesion, followed by administration of one or more doses of the MAGE-A4 TCR of the present invention. In some embodiments, radiation therapy may be administered locally to a tumor lesion, followed by systemic administration of the MAGE-A4 TCR of the present invention, to enhance the local immunogenicity of the patient's tumor (active radiation) and / or kill tumor cells (ablative radiation).

[0175] The additional therapeutically active agent / component may be administered prior to, simultaneously with, or following administration of the MAGE-A4 TCR of the present invention, and for purposes of this disclosure, such a dosing regimen will be considered administration of the MAGE-A4 TCR "in combination" with the second therapeutically active component.

[0176] The additional therapeutically active ingredient may be administered to the subject prior to administration of the MAGE-A4 TCR of the present invention. The TCR may be administered after administration. In yet other embodiments, the additional therapeutically active component may be administered to the subject simultaneously with administration of the MAGE-A4 TCR of the present invention. "Concurrent" administration, for purposes of the present invention, includes, for example, administering the MAGE-A4 TCR and the additional therapeutically active component to the subject in a single dosage form (e.g., co-formulated) or in separate dosage forms that are administered to the subject within about 30 minutes of each other. When administered in separate dosage forms, each dosage form may be administered via the same route, or alternatively, each dosage form may be administered via a different route. In any event, administration of the components in a single dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes are all considered "concurrent administration" for purposes of this disclosure. For purposes of this disclosure, administration of the MAGE-A4 TCR "before," "concurrently," or "after" (as these terms are defined above) the additional therapeutically active component is considered administration of the MAGE-A4 TCR "in combination with" the additional therapeutically active component.

[0177] The present invention is further illustrated by the following examples, which are not intended to be limiting in any way. The entire contents of all references, patents, and published patent applications cited throughout this application, as well as any figures, are hereby incorporated by reference. [Example]

[0178] Example 1. Identification of MAGE-A4-specific T cell receptors Veloci-T® mice, which are mice humanized for cellular immune system components (see, e.g., PCT Publication WO2016 / 164492, the entire contents of which are incorporated herein by reference), were immunized with the MAGE-A4 (286-294) peptide (KVLEHVVRV, SEQ ID NO: 609), specifically presented by human HLA-A2, diluted in PBS and mixed with an equal volume of adjuvant, e.g., complete Freund's adjuvant (CFA, Chondrex, Inc.). Spleen suspensions were obtained from immunized mice and dissociated. Erythrocytes were lysed in ACK lysis buffer (Life Technologies), and splenocytes were suspended in RPMI complete medium. Isolated splenocytes were sorted, and single T cells that bound the MAGE-A4 (286-294) peptide in the context of MHC were isolated by fluorescence-activated cell sorting (FACS). Isolated T cells were seeded into single-well plates and mixed with TCR alpha and beta variable region-specific PCR primers. cDNA from each single T cell was synthesized via reverse transcriptase (RT). Each resulting RT product was then divided and transferred to two corresponding wells for subsequent TCR beta and alpha PCR. One set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific to the TCR beta variable region leader sequence or a 5' degenerate primer specific to the TCR alpha chain variable region leader sequence and a 3' primer specific to the TCR constant region to generate an amplicon. It was then amplified again by PCR using a 5' degenerate primer specific to the TCR beta variable region framework 1 or a 5' degenerate primer specific to the TCR alpha chain variable region framework 1 and a 3' primer specific to the TCR constant region to generate an amplicon for cloning. The PCR products derived from TCR beta and alpha were cloned into expression vectors containing the beta and alpha constant regions, respectively. Expression vectors expressing full-length beta and alpha chain pairs were transfected into CHO cells and tested for binding with a commercially available MAGE-A4 / HLA tetramer reagent (HLA-A02:01 MAGE-A4 tetramer, MBL International Corporation).CHO cells were incubated with soluble HLA-A2(KVLEHVVRV) (SEQ ID NO: 609) tetramer and an antibody specific for the mouse TCR constant region (clone H57-597) (Biolegend, San Diego, CA). Samples were then analyzed on an LSRFortessa X-20 (BD Biosciences, San Jose, CA). To calculate the percentage of tetramer-positive cells, antigen-positive (Ag+) gating was set based on a negative control TCR that does not bind to the HLA-A2(KVLEHVVRV) (SEQ ID NO: 609) tetramer using FlowJo (LLC, Ashland, OR). All Ag+ TCRs met the FlowJo criteria of ≥1% of cells, with Ag+ gating by a mean fluorescence intensity (MFI) >250. Ag+ TCRs were determined by next-generation sequencing, and the total number of TCRs expressing identical TCR alpha and beta nucleotide sequences is shown in Table 1 below. [Table 1-1] [Table 1-2]

[0179] A detailed list of the TCR beta chain variable domain CDR1, CDR2, and CDR3 amino acid sequences and the alpha chain variable domain CDR1, CDR2, and CDR3 amino acid sequences determined above is provided in Table 2. A detailed list of the TCR beta chain variable domain CDR1, CDR2, and CDR3 polynucleic acid sequences and the alpha chain variable domain CDR1, CDR2, and CDR3 polynucleic acid sequences determined above is provided in Table 3. Table 4 provides the amino acid and nucleotide sequences of the TCR beta chain variable region and alpha chain variable region. [Table 2-1] [Table 2-2] [Table 3-1] Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 4-1 Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 4-6 Table 4-7 Table 4-8 Table 4-9 Table 4-10 Table 4-11 Table 4-12 [Table 4-13] [Table 4-14] [Table 4-15] [Table 4-16] [Table 4-17] [Table 4-18] [Table 4-19] [Table 4-20]

[0180] Example 2. Identification of MAGE-A4-specific T cell receptors Veloci-T® mice, which are mice humanized for cellular immune system components (see, e.g., PCT Publication WO2016 / 164492, the entire contents of which are incorporated herein by reference), were immunized with the MAGE-A4(230-239) peptide (GVYDGREHTV, SEQ ID NO: 612), specifically presented by human HLA-A2, diluted in PBS and mixed with an equal volume of adjuvant, e.g., complete Freund's adjuvant (CFA, Chondrex, Inc.). Spleen suspensions were obtained from immunized mice and dissociated. Erythrocytes were lysed in ACK lysis buffer (Life Technologies), and splenocytes were suspended in RPMI complete medium. Isolated splenocytes were sorted, and single T cells that bound the MAGE-A4(230-239) peptide in the context of MHC were isolated by fluorescence-activated cell sorting (FACS). Isolated T cells were seeded into single-well plates and mixed with TCR alpha and beta variable region-specific PCR primers. cDNA from each single T cell was synthesized via reverse transcriptase (RT) reaction. The resulting RT products were then divided and transferred to two corresponding wells for subsequent TCR beta and alpha PCR. One set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific to the TCR beta variable region leader sequence, or a 5' degenerate primer specific to the TCR alpha chain variable region leader sequence, and a 3' primer specific to the TCR constant region to generate amplicons. Next, the PCR products were amplified again by PCR using a 5' degenerate primer specific to the TCR beta variable region framework 1, or a 5' degenerate primer specific to the TCR alpha chain variable region framework 1, and a 3' primer specific to the TCR constant region to generate amplicons for cloning. The TCR beta and alpha-derived PCR products were cloned into expression vectors containing the beta and alpha constant regions, respectively.Expression vectors expressing full-length beta and alpha chain pairs were transfected into CHO cells and tested for binding to a commercially available MAGE-A4 / HLA tetramer reagent (HLA-A02:01 MAGE-A4 tetramer, MBL International Corporation) or a commercially available MAGE-A8 / HLA tetramer reagent (HLA-A02:01 MAGE-A8 tetramer, MBL International Corporation). CHO cells were incubated with an antibody specific for the mouse TCR constant region (clone H57-597) (Biolegend, San Diego, CA) and either soluble HLA-A2 MAGE-A4 (GVYDGREHTV) (SEQ ID NO: 612) tetramer or soluble HLA-A2 MAGE-A8 (GLYDGREHSV) (SEQ ID NO: 613). Samples were then analyzed using an LSRFortessa X-20 (BD Biosciences, San Jose, CA). To calculate the percentage of tetramer-positive cells for MAGE-A4 (230–239), antigen-positive (Ag+) gating was based on a negative control TCR that does not bind to the HLA-A2 (GVYDGREHTV) (SEQ ID NO: 612) tetramer. To calculate the percentage of tetramer-positive cells for MAGE-A8, antigen-positive (Ag+) gating was based on a negative control TCR that does not bind to the HLA-A2 (GLYDGREHSV) (SEQ ID NO: 613) tetramer using FlowJo (LLC, Ashland, OR). All Ag+ TCRs met the FlowJo criterion of ≥1% of cells with Ag+ gating by a mean fluorescence intensity (MFI) >250. However, a more stringent cutoff, such as 3%, could be used to isolate Ag+ TCRs that performed better in the binding assay. Ag+ TCRs were determined by next-generation sequencing.Table 5 shows the total number of TCRs expressing the same TCR alpha and beta nucleotide sequences, the % tetramers in Ag+ gating (MAGE-A4(230-239)), the % tetramers in Ag+ gating (MAGE-A8(232-241)), and the ratio of the latter two values ​​(% tetramers in Ag+ gating (MAGE-A4(230-239)) divided by the % tetramers in Ag+ gating (MAGE-A8(232-241))). This ratio is an indicator of the specificity of MAGE-A4(230-239) binding. [Table 5-1] [Table 5-2]

[0181] A detailed list of the TCR beta chain variable CDR1, CDR2, and CDR3 amino acid sequences and alpha chain variable domain CDR1, CDR2, and CDR3 amino acid sequences determined as above is provided in Table 6, and the corresponding nucleic acid sequences are provided in Table 7. Table 8 provides the amino acid and nucleotide sequences of the TCR beta chain variable region and alpha chain variable region.

[0182] Table 9 provides the TCR gene families of the isolated TCR alpha and beta variable regions and joining regions, and Table 10 provides the amino acid and polynucleic acid sequence identifiers of the alpha and beta variable chains and CDRs. [Table 6] [Table 7-1] [Table 7-2] [Table 7-3] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] [Table 9] [Table 10]

[0183] Example 3. Cytotoxicity of MAGE-A4 TCR against A375 melanoma cells This example illustrates the ability of certain Mage-A4 TCRs of the invention to selectively kill A375 melanoma cells.

[0184] method T cell manipulation Pan T cells were purified by negative selection from human peripheral blood mononuclear cells (PBMCs) using the EasySep Human T Cell Isolation Kit (StemCell Technologies 17951) and then cryopreserved. On day 0 of the experiment, T cells were thawed and cultured at 1 × 10 cells in medium (CTS OpTmizer medium [Life Technologies A1048501] supplemented with 10 mg / ml gentamicin, 4 mM L-glutamine) containing 200 U / ml human IL-2 and anti-CD3 / anti-CD28 activation beads at a 1:1 bead:cell ratio (Life Technologies 111.32D). 6 On day 3, the activated beads were removed and 5 × 10 cells were transfected with 20 mg of Cas9 protein (Life Technologies A36499) complexed with sgRNAs targeting the TRAC and TRBC loci using a Nucleofector IIb device and a human T cell nucleofection kit (Lonza VVPA-1002). 6 After nucleofection, cells were transduced with adeno-associated virus (AAV) vectors engineered to target TCRa and TCRb expression cassettes to the human TRAC locus. Every 2–3 days, cells were transfected at approximately 1 × 10 6 At various times after transduction of the TCR constructs, cells were stained with antibodies against surface markers and peptide-MHC tetramer reagents and then analyzed by flow cytometry to quantify transduction efficiency.

[0185] Cytotoxicity assay Antigen-specific cytotoxic T cell function was assessed by calcein release assay. Target tumor cells were labeled in the presence of 8 mM calcein AM dye (Life Technologies C1430) at 37°C for 30 minutes and then washed with medium. 1 × 10 4Target cells were seeded per assay well in a 96-well plate with various dilutions of transduced T cells or untransduced (UTD) controls. After 2 hours, released fluorescent dye was measured in the culture supernatant. Spontaneous release (SR) was measured in wells containing labeled target cells but no T cells, and maximum release (MR) was measured in wells with labeled target cells in the presence of 0.5% Triton X-100. The percent specific cytotoxicity was determined as 100×(test release - SR) / (MR - SR).

[0186] result Primary human T cells were engineered to express MAGE-A4-specific TCRs against two HLA-A2-restricted peptides (MAGE-A4 286-294 (KVLEHVVRV, SEQ ID NO: 609) and MAGE-A4 230-239 (GVYDGREHTV, SEQ ID NO: 294)), or an unrelated HLA-A2-restricted peptide derived from a viral protein (HPV). As shown in Figure 1, flow cytometry analysis using peptide-MHC tetramer reagents confirmed the expression and predicted antigen specificity of the transduced TCRs.

[0187] To verify the cytotoxic activity of the TCRs, engineered T cells were tested for their ability to direct cytolytic function against MAGE-A4-expressing tumor cells in calcein AM dye-release assays, performed in duplicate (Figures 2A, 2B, 3A, and 3B). These assays confirmed that MAGE-A4-specific TCRs (against both the MAGE-A4 286-294 and MAGE-A4 230-239 peptides) mediated killing of A375 melanoma cells in a dose-dependent manner, whereas TCRs against an unrelated HLA-A2-restricted peptide did not.

[0188] Example 4. MAGE-A4(286-294) TCR in Jurkat Cell TCR Signaling Bioassay This example illustrates the ability of selected Mage-A4 TCRs of the invention to activate TCR signaling in Jurkat cells.

[0189] method Jurkat cell line generation Jurkat cell lines lacking endogenous TCRα and TCRβ expression were generated by knockout of these genes and then engineered to allow single-copy Cre recombinase-mediated insertion of transgenic TCR constructs. An AP1 response element-driven luciferase reporter was then integrated into this parent bioassay line. TCR bioassay lines were generated by Cre-mediated insertion of customized TCRα / β expression constructs.

[0190] TCR activation bioassay Jurkat bioassay lines expressing the TCR constructs were FACS-sorted for homogeneity and then tested in a peptide-MHC stimulation assay. 293T cells (HLA-A2*01) were seeded into assay wells with various dilutions of antigen (MAGE-A4 286-294 (KVLE)) or an irrelevant peptide (MAGE-A4 230-239 (GVY)). After 2 hours of incubation, engineered Jurkat cells were added to the wells at a 3:1 Jurkat cell:293T cell ratio and incubated for an additional 5 hours. Luciferase reporter activity was determined by measuring luminescence output in the assay cells.

[0191] result Jurkat bioassay lines engineered to carry an AP1 response element-driven luciferase transgene and express a MAGE-A4-specific TCR or a TCR against an irrelevant antigen (NY-ESO-1) were tested in peptide stimulation assays. As shown in Figure 4A and Figure 4B, TCRs specific for HLA-A2*01-restricted MAGE-A4 (286-294 (KVLE)) were reactive only to the cognate (KVLE) peptide, but not to the irrelevant MAGE-A4 (230-239 (GVY)) peptide. Parental TCR-negative (TCR-) cells and cells expressing an irrelevant TCR (against NY-ESO-1) showed no reactivity. In addition, Figure 4A and Figure 4B show that AP1 reporter activation induced by MAGE-A4-specific TCRs was dose-dependent.

[0192] Example 5. Specific activity of MAGE-A4 (230-239) TCR in T cells This example illustrates the ability of selected Mage-A4 TCRs of the invention to activate TCR signaling in T cells.

[0193] Human peripheral blood mononuclear cells (PBMCs) (n = 3 donors) were activated and transduced with a lentiviral vector encoding a TCR that binds to the HLA-A2-restricted MAGE-A4 (230-239 (GVY)) and expanded in vitro for 10 days before freezing. After thawing, T cells containing the above TCR were cultured at a 1:1 ratio with T2 cells pulsed with the MAGE-A4 230-239 peptide. Reactivity was measured as interferon-γ release 24 hours after coculture. T cells alone were used as a control for antigen-independent interferon-γ release. T cells cultured with unpulsed T2 cells or T2 cells pulsed with an irrelevant peptide were used as controls for specificity. Figure 5 shows the mean + / - standard error of the mean (SEM) of interferon-γ signals from n=3 donors and demonstrates that the MAGE-A4(230-239)-specific TCR of the present disclosure exhibits specific activity against T2 cells pulsed with MAGE-A4(230-239) peptide.

[0194] To test activity against tumor cells expressing MAGE-A4, human PBMCs (n = 3 donors) were activated and transduced with a lentiviral vector encoding the MAGE-A4(230-239) TCR, expanded in vitro for 10 days, and then frozen. After thawing, MAGE-A4(230-239) TCR T cells were cultured at a 5:1 ratio with tumor cells expressing manipulated levels of HLA-A2 and MAGE-A4 (A549.A2.MAGEA4 cells) or endogenous levels of HLA-A2 and MAGE-A4 (A375 cells). Reactivity was measured as interferon-γ release 24 hours after coculture. T cells alone were used as a control for antigen-independent interferon-γ release. T cells cultured with unmodified A549 cells (MAGE-A4-negative and HLA-A2-negative) or A549 cells modified to express HLA-A2 (A549.A2) were used as specificity controls. Figure 6 shows the mean + / - standard error of the mean (SEM) of interferon-γ signals from n=3 donors, demonstrating that the disclosed MAGE-A4 (230-239)-specific TCR exhibits specific activity against tumor cells expressing HLA-A2 and MAGE-A4. In a separate assay, T cells were cocultured with A549 cells, A549.A2 cells, NCI-H520 (HLA-A2-negative / MAGE-A4-positive) cells, and MCF-7 (HLA-A2-positive / MAGE-A4-negative) cells to provide an additional control for specificity against the HLA-A2 / MAGE-A4 complex. Figure 7 shows the mean + / - standard error of the mean (SEM) of interferon-γ signals from n=3 donors, demonstrating that the disclosed MAGE-A4(230-239)-specific TCR exhibits specific activity against tumor cells expressing HLA-A2 and MAGE-A4. In a further assay, MAGE-A4(230-239) TCR-expressing T cells were cultured at a 5:1 ratio with tumor cells expressing low or moderate endogenous levels of MAGE-A4 (U2-OS and NCI-H1703, respectively). Reactivity was measured as IFNγ release after 24 hours of coculture.Figure 8 shows the mean + / - standard error of the mean (SEM) of interferon gamma signals from n=3 donors and demonstrates that the MAGE-A4(230-239)-specific TCR of the present disclosure exhibits specific activity against HLA-A2-expressing tumor cells that express only low or moderate endogenous levels of MAGE-A4.

[0195] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims. The contents of all references, patents, and published patent applications cited throughout this application are hereby incorporated by reference. The present invention provides, for example, the following items. (Item 1) 1. A T cell receptor (TCR) that specifically binds to an HLA-A2-presented cancer-testis antigen melanoma associated antigen 4 (MAGE-A4) peptide comprising the amino acid sequence of KVLEHVVRV (SEQ ID NO: 609) (MAGE-A4 286-294), wherein the TCR comprises an alpha chain variable domain comprising a complementarity determining region (CDR) 3, and wherein the CDR3 is represented by Formula I: N1-N2-N3-N4-N5-N6-N7-N8-N9-N 10 -N 11 -N 12 -N 13 -N 14 -N 15 (Formula I) comprising the amino acid sequence wherein N1 is a nonpolar amino acid; N2 is Val, but may be present or absent; N3 is Tyr, Gly, Leu, Val, Glu, Met, Ala, or Phe; N4 is Arg, Glu, Ser, Asn, Gln, Lys, Asp, Gly, or Met, but may be present or absent; N5 is Ser, Arg, Glu, Leu, Ala, Asp, Pro, Met, Gly, or Lys, but may be present or absent; N6 is Ala, Asp, Gly, Ser, Val, Pro, Leu, Tyr, or Thr, but may be present or absent; N7 is Thr, Pro, Ser, Glu, Asp, Trp, Arg, Asn, Ile, Gln, or Leu; N8 is His, Trp, Thr, Lys, Tyr, or Ala; N9 is Asn, Gly, Lys, Ile, Ser, or Arg; N 10 is Gln, Lys, Gly, Thr, Leu, Asp, or Ser, but may be present or absent; N 11 is Phe, Asn, Thr, Tyr, Ala, Leu, Met, or Glu, but may be present or absent; N 12 is Lys, Phe, Tyr, or Asp, but may be present or absent; N 13 is Lys or Gly, but may be present or absent, N 14 is Thr, Leu, or Tyr, but may be present or absent; N 15 is Tyr, Gln, Ile, Thr, Val, or Arg, T cell receptor (TCR). (Item 2) 2. The TCR of item 1, wherein N1 is Ala, Ile, or Gly. (Item 3) 1. A T cell receptor (TCR) that specifically binds to an HLA-A2-presented cancer-testis antigen melanoma associated antigen 4 (MAGE-A4) peptide comprising the amino acid sequence of KVLEHVVRV (SEQ ID NO: 609) (MAGE-A4 286-294), wherein the TCR comprises a beta chain variable domain comprising a complementarity determining region (CDR) 3, and wherein the CDR3 is represented by Formula II: N1-N2-N3-N4-N5-N6-N7-N8-N9-N 10 -N 11 -N 12 -N 13 -N 14 -N 15 -N 16 -N 17 -N 18 (Formula II) comprising the amino acid sequence wherein N1 is Ala or Ser; N2 is Ala, Ser, or Thr; N3 is Ser, Gly, or Trp; N4 is Leu, Tyr, Trp, Asp, Phe, Gly, Pro, or His; N5 is Gly or Asp, but may be present or absent; N6 is Phe or Arg, but may be present or absent; N7 is Trp, Phe, Asp, Pro, Tyr, Gly, Thr, Ser, or Val, but may be present or absent; N8 is Pro, Arg, Asp, Tyr, Gln, Asn, or Gly, but may be present or absent; N9 is Asp but may be present or absent; N 10 is an Arg, which may or may not be present, N 11 is Gly, Ala, or Thr, but may be present or absent; N 12 is Ser, Trp, Thr, Gly, Val, Leu, Arg, Met, Tyr, or Gln, N 13 is Gly, but may be present or absent, N 14 is Asn, Asp, Gly, Thr, Pro, Gln, or His, but may be present or absent; N 15is Thr, Ser, Glu, Asn, Tyr, Gln, Asp, or Pro, but may be present or absent; N 16 is Glu, Pro, Lys, Thr, Ala, Gly, or Gln, but may be present or absent; N 17 is Ala, Leu, Ile, Tyr, or Gln, but may be present or absent; N 18 is Phe, His, Tyr, or Thr, T cell receptor (TCR). (Item 4) The TCR of item 1 or 2, wherein the alpha chain variable domain further comprises CDR1 and CDR2, wherein the CDR1 comprises any one of the alpha chain variable domain CDR1 amino acid sequences shown in Table 2, and the CDR2 independently comprises any one of the alpha chain variable domain CDR2 amino acid sequences shown in Table 2. (Item 5) The TCR of item 3, wherein the beta chain variable domain further comprises CDR1 and CDR2, wherein the CDR1 comprises any one of the beta chain variable CDR1 amino acid sequences shown in Table 2, and the CDR2 independently comprises any one of the beta chain variable domain CDR2 amino acid sequences shown in Table 2. (Item 6) 6. The TCR of any one of items 1 to 5, wherein the TCR comprises at least one TCR alpha chain variable domain and / or at least one beta chain variable domain. (Item 7) 7. The TCR of item 6, wherein the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain. (Item 8) A TCR described in any one of items 3 to 7, comprising an alpha chain variable domain CDR1, CDR2, and CDR3 contained within any one of the alpha chain variable domain sequences listed in Table 4, and a beta chain variable domain CDR1, CDR2, and CDR3 contained within any one of the beta chain variable domain sequences listed in Table 4. (Item 9) The TCR according to any one of items 1 to 8, comprising an alpha chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the alpha chain variable domain amino acid sequences listed in Table 4. (Item 10) 10. The TCR of any one of items 1 to 9, comprising a beta chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 4. (Item 11) 11. The TCR of any one of items 1 to 10, comprising: (a) an alpha chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 4; and (b) a beta chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta chain variable domain amino acid sequences listed in Table 4. (Item 12) (a) an alpha chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337, 353, 369, 385, 401, 417, 433, 449, 465, 481, 497, 513, 529, 545, 561, 577, and 593; (b) an alpha chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 466, 482, 498, 514, 530, 546, 562, 578, and 594; (c) an alpha chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307, 323, 339, 355, 371, 387, 403, 419, 435, 451, 467, 483, 499, 515, 531, 547, 563, 579, and 595; (d) a beta chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, 329, 345, 361, 377, 393, 409, 425, 441, 457, 473, 489, 505, 521, 537, 553, 569, 585, and 601; (e) a beta chain variable domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554, 570, 586, and 602; (f) a beta chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379, 395, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555, 571, 587, and 603. (Item 13) Sequence numbers 7 / 15, 23 / 31, 39 / 47, 55 / 63, 71 / 79, 87 / 95, 103 / 111, 119 / 127, 135 / 143, 151 / 159, 167 / 175, 183 / 191, 199 / 207, 215 / 223, 231 / 239, 247 / 255, 263 / 271, 279 / 287, 295 / 303, 311 / 319, 327 / 335, 343 / 351, 359 / 367, 37 13. The TCR of item 12, comprising an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of: 5 / 383, 391 / 399, 407 / 415, 423 / 431, 439 / 447, 455 / 463, 471 / 479, 487 / 495, 503 / 511, 519 / 527, 535 / 543, 551 / 559, 567 / 575, 583 / 591, and 599 / 607. (Item 14) SEQ ID NOs: 87 / 31, 23 / 95, 231 / 607, 231 / 223, 231 / 591, 231 / 255, 231 / 271, 231 / 79, 231 / 47, 231 / 399, 599 / 239, 599 / 223, 599 / 591, 599 / 255, 599 / 271, 599 / 79 , 599 / 47, 599 / 399, 215 / 239, 215 / 607, 215 / 591, 215 / 255, 215 / 271, 215 / 79, 215 / 47, 215 / 399, 583 / 239, 583 / 607, 583 / 223, 583 / 255, 583 / 271, 583 / 79, 583 / 47, 583 / 399, 247 / 239, 247 / 607, 247 / 223, 247 / 591, 247 / 271, 247 / 79, 247 / 47, 247 / 399, 263 / 239, 263 / 607, 263 / 223, 263 / 591, 263 / 255, 263 / 79, 2 63 / 47, 263 / 399, 71 / 239, 71 / 607, 71 / 223, 71 / 591, 71 / 255, 71 / 271, 71 / 47, 71 / 399, 39 / 239, 39 / 607, 39 / 223, 39 / 591, 39 / 255, 39 / 271, 39 / 79, 39 / 399, 3 91 / 239, 391 / 607, 391 / 223, 391 / 591, 391 / 255, 391 / 271, 391 / 79, 391 / 47, 439 / 127, 439 / 319, 439 / 287, 439 / 15, 439 / 111, 439 / 383, 439 / 191, 439 / 511, 439 / 527, 439 / 559, 439 / 207, 119 / 447, 119 / 319, 119 / 287, 119 / 15, 119 / 111, 119 / 383, 119 / 191, 119 / 511, 119 / 527, 119 / 559, 119 / 207, 311 / 447, 311 / 12 7, 311 / 287, 311 / 15, 311 / 111, 311 / 383, 311 / 191, 311 / 511, 311 / 527, 311 / 559, 311 / 207, 279 / 447, 279 / 127, 279 / 319, 279 / 15, 279 / 111, 279 / 383, 279 / 1 91, 279 / 511, 279 / 527, 279 / 559, 279 / 207, 7 / 447, 7 / 127, 7 / 319, 7 / 287, 7 / 111, 7 / 383, 7 / 191, 7 / 511, 7 / 527, 7 / 559, 7 / 207, 103 / 447, 103 / 127, 103 / 319,103 / 287, 103 / 15, 103 / 383, 103 / 191, 103 / 511, 103 / 527, 103 / 559, 103 / 207, 375 / 447, 375 / 127, 375 / 319, 375 / 287, 375 / 15, 375 / 111, 375 / 191, 375 / 511, 375 / 527, 375 / 559, 375 / 207, 183 / 447, 183 / 127, 183 / 319, 183 / 287, 183 / 15, 183 / 111, 183 / 383, 183 / 511, 183 / 527, 183 / 559, 183 / 207, 503 / 447, 503 / 127, 503 / 319, 503 / 287, 503 / 15, 503 / 111, 503 / 383, 503 / 191, 503 / 527, 503 / 559, 503 / 20 7, 519 / 447, 519 / 127, 519 / 319, 519 / 287, 519 / 15, 519 / 111, 519 / 383, 519 / 191, 519 / 511, 519 / 559, 519 / 207, 551 / 447, 551 / 127, 551 / 319, 551 / 287, 551 / 15, 551 / 111, 551 / 383, 551 / 191, 551 / 511, 5 13. The TCR of item 12, comprising an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of 51 / 527, 551 / 207, 199 / 447, 199 / 127, 199 / 319, 199 / 287, 199 / 15, 199 / 111, 199 / 383, 199 / 191, 199 / 511, 199 / 527, and 199 / 559. (Item 15) (Item 16) A TCR that competes for binding with the TCR according to any one of Items 1 to 14. 16. The TCR of any one of items 1 to 15, further comprising a detectable moiety. (Item 17) 17. A pharmaceutical composition comprising the TCR according to any one of items 1 to 16 and a pharmaceutically acceptable carrier or diluent. (Item 18) An isolated cell presenting the TCR according to any one of items 1 to 16. (Item 19) 17. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the alpha chain variable domain of the TCR according to any one of items 1 to 16. (Item 20) 17. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the beta chain variable domain of the TCR according to any one of items 1 to 16. (Item 21) 21. A vector comprising the polynucleotide molecule of item 19 or 20. (Item 22) 22. An isolated cell expressing the vector of item 21. (Item 23) A method for treating a subject having a MAGE-A4-related disease or disorder, comprising administering to the subject a therapeutically effective amount of the TCR of any one of items 1 to 16, the pharmaceutical composition of item 17, or a plurality of isolated cells of item 18, thereby treating the subject. (Item 24) 24. The method of claim 23, wherein the MAGE-A4-associated disease or disorder is a MAGE-A4-associated cancer. (Item 25) The MAGE-A4-associated cancers are liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, metastatic solid tumor, colon cancer, gastric cancer 25. The method of claim 24, wherein the cancer is gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, or recurrent non-small cell lung cancer. (Item 26) 26. The method of any one of items 23 to 25, wherein the TCR, the pharmaceutical composition, or the plurality of cells is administered to the subject in combination with a second therapeutic agent. (Item 27) 27. The method of any one of items 23 to 26, wherein the TCR, the pharmaceutical composition, or the plurality of cells is administered to the subject subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. (Item 28) 1. A polynucleotide molecule encoding a T cell receptor (TCR), wherein the TCR specifically binds to an HLA-A2-presented cancer-testis antigen melanoma associated antigen 4 (MAGE-A4) peptide comprising the amino acid sequence of KVLEHVVRV (SEQ ID NO: 609) (MAGE-A4 286-294), wherein the TCR has a property selected from the group consisting of: (a) not binding to cells expressing predicted off-target peptides, as determined by a luminescence assay; (b) activating T cell responses to a greater extent than patient-derived MAGE-A4-specific TCRs, as determined by a TCR-mediated T cell signaling luminescence bioassay; and (c) activating T cell responses to a greater extent than affinity-matured (e.g., phage-displayed) MAGE-A4-specific TCRs, as determined by a TCR-mediated T cell signaling luminescence bioassay. (Item 29) 29. The polynucleotide molecule of item 28, encoding at least one TCR alpha chain variable domain and / or at least one beta chain variable domain. (Item 30) 29. The polynucleotide molecule of item 28, wherein the TCR comprises an alpha chain variable domain complementarity determining region (CDR) 1, CDR2, and CDR3 contained within any one of the alpha chain variable domain sequences listed in Table 4, and a beta chain variable domain CDR1, CDR2, and CDR3 contained within any one of the beta chain variable domain sequences listed in Table 4. (Item 31) 31. The polynucleotide molecule of item 29 or 30, wherein the TCR comprises an alpha chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 4. (Item 32) 32. The polynucleotide molecule of any one of Items 28 to 31, wherein the TCR comprises a beta chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 4. (Item 33) 33. The polynucleotide molecule of any one of Items 28 to 32, wherein the TCR comprises: (a) an alpha chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the alpha chain variable domain amino acid sequences listed in Table 4; and (b) a beta chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 4. (Item 34) The TCR (a) an alpha chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337, 353, 369, 385, 401, 417, 433, 449, 465, 481, 497, 513, 529, 545, 561, 577, and 593; (b) an alpha chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 466, 482, 498, 514, 530, 546, 562, 578, and 594; (c) an alpha chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307, 323, 339, 355, 371, 387, 403, 419, 435, 451, 467, 483, 499, 515, 531, 547, 563, 579, and 595; (d) a beta chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, 329, 345, 361, 377, 393, 409, 425, 441, 457, 473, 489, 505, 521, 537, 553, 569, 585, and 601; (e) a beta chain variable domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554, 570, 586, and 602; (f) a beta chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379, 395, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555, 571, 587, and 603. (Item 35) The TCR is selected from the group consisting of SEQ ID NOs: 7 / 15, 23 / 31, 39 / 47, 55 / 63, 71 / 79, 87 / 95, 103 / 111, 119 / 127, 135 / 143, 151 / 159, 167 / 175, 183 / 191, 199 / 207, 215 / 223, 231 / 239, 247 / 255, 263 / 271, 279 / 287, 295 / 303, 311 / 319, 327 / 335, 343 / 351, 359 / 367, 37 35. The polynucleotide molecule of item 34, comprising an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of 5 / 383, 391 / 399, 407 / 415, 423 / 431, 439 / 447, 455 / 463, 471 / 479, 487 / 495, 503 / 511, 519 / 527, 535 / 543, 551 / 559, 567 / 575, 583 / 591, and 599 / 607. (Item 36) The TCR is selected from the group consisting of SEQ ID NOs: 87 / 31, 23 / 95, 231 / 607, 231 / 223, 231 / 591, 231 / 255, 231 / 271, 231 / 79, 231 / 47, 231 / 399, 599 / 239, 599 / 223, 599 / 591, 599 / 255, 599 / 271, 599 / 79, 599 / 47, 599 / 399, 215 / 239, 215 / 607, 215 / 591, 215 / 255, 215 / 271, 215 / 79, 215 / 47, 215 / 399, 583 / 239, 583 / 607, 583 / 223, 583 / 255, 583 / 271, 583 / 79, 583 / 47, 583 / 399, 247 / 239, 247 / 607, 247 / 223, 247 / 591, 247 / 271, 247 / 79, 247 / 47, 247 / 399, 263 / 239, 263 / 607, 263 / 223, 263 / 591, 263 / 255, 263 / 79, 263 / 47, 263 / 399, 71 / 239, 71 / 607, 71 / 223, 71 / 591, 71 / 255, 71 / 271, 71 / 47, 71 / 399, 39 / 239, 39 / 607, 39 / 223, 39 / 591, 39 / 255, 39 / 271, 39 / 79, 39 / 399, 391 / 239, 391 / 607, 391 / 223, 391 / 591, 391 / 255, 391 / 271, 391 / 79, 391 / 47, 439 / 127, 439 / 319, 439 / 287, 439 / 15, 439 / 111, 439 / 383, 439 / 191, 439 / 511, 439 / 527, 439 / 559, 439 / 207, 119 / 447, 119 / 319, 119 / 287, 119 / 15, 119 / 111, 119 / 383, 119 / 191, 119 / 511, 119 / 527, 119 / 559, 119 / 207, 311 / 44 7, 311 / 127, 311 / 287, 311 / 15, 311 / 111, 311 / 383, 311 / 191, 311 / 511, 311 / 527, 311 / 559, 311 / 207, 279 / 447, 279 / 127, 279 / 319, 279 / 15, 279 / 111, 279 / 3 83, 279 / 191, 279 / 511, 279 / 527, 279 / 559, 279 / 207, 7 / 447, 7 / 127, 7 / 319, 7 / 287, 7 / 111, 7 / 383, 7 / 191, 7 / 511, 7 / 527, 7 / 559, 7 / 207, 103 / 447, 103 / 127,103 / 319, 103 / 287, 103 / 15, 103 / 383, 103 / 191, 103 / 511, 103 / 527, 103 / 559, 103 / 207, 375 / 447, 375 / 127, 375 / 319, 375 / 287, 375 / 15, 375 / 111, 375 / 191, 375 / 511, 375 / 527, 375 / 559, 375 / 207, 183 / 44 7, 183 / 127, 183 / 319, 183 / 287, 183 / 15, 183 / 111, 183 / 383, 183 / 511, 183 / 527, 183 / 559, 183 / 207, 503 / 447, 503 / 127, 503 / 319, 503 / 287, 503 / 15, 503 / 111, 503 / 383, 503 / 191, 503 / 527, 503 / 559, 503 / 2 07, 519 / 447, 519 / 127, 519 / 319, 519 / 287, 519 / 15, 519 / 111, 519 / 383, 519 / 191, 519 / 511, 510 / 559, 519 / 207, 551 / 447, 551 / 127, 551 / 319, 551 / 287, 551 / 15, 551 / 111, 551 / 383, 551 / 191, 551 / 511, 551 / 35. The polynucleotide molecule of item 34, comprising an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of: 527, 551 / 207, 199 / 447, 199 / 127, 199 / 319, 199 / 287, 199 / 15, 199 / 111, 199 / 383, 199 / 191, 199 / 511, 199 / 527, and 199 / 559. (Item 37) The TCR (a) an alpha chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452, 468, 484, 500, 516, 532, 548, 564, 580, and 596; (b) an alpha chain variable domain CDR2 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5, 21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261, 277, 293, 309, 325, 341, 357, 373, 389, 405, 421, 437, 453, 469, 485, 501, 517, 533, 549, 565, 581, and 597; (c) an alpha chain variable domain CDR3 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 470, 486, 502, 518, 534, 550, 566, 582, and 598; (d) a beta chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 460, 476, 492, 508, 524, 540, 556, 572, 588, and 604; (e) a beta chain variable domain CDR2 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285, 301, 317, 333, 349, 365, 381, 397, 413, 429, 445, 461, 477, 493, 509, 525, 541, 557, 573, 589, and 605; (f) a beta chain variable domain CDR3 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, ​​398, 414, 430, 446, 462, 478, 494, 510, 526, 542, 558, 574, 590, and 606. (Item 38) The TCR is selected from the group consisting of SEQ ID NOs: 8 / 16, 24 / 32, 40 / 48, 56 / 64, 72 / 80, 88 / 96, 104 / 112, 120 / 128, 136 / 144, 152 / 160, 168 / 176, 184 / 192, 200 / 208, 216 / 224, 232 / 240, 248 / 256, 264 / 272, 280 / 288, 296 / 304, 312 / 320, 328 / 336, 344 / 352, 360 / 368 38. The polynucleotide molecule of claim 37, comprising an alpha chain variable domain / beta chain variable domain nucleic acid sequence pair selected from the group consisting of: 376 / 384, 392 / 400, 408 / 416, 424 / 432, 440 / 448, 456 / 464, 472 / 480, 488 / 496, 504 / 512, 520 / 528, 536 / 544, 552 / 560, 568 / 576, 584 / 592, 600 / 608. (Item 39) A vector comprising the polynucleotide molecule according to any one of Items 28 to 38. (Item 40) 39. An isolated cell comprising the vector of item 39. (Item 41) 41. A method of treating a subject having a MAGE-A4-related disease or disorder, comprising administering to the subject a plurality of cells according to item 40, thereby treating the subject. (Item 42) 42. The method of claim 41, wherein the MAGE-A4-associated disease or disorder is a MAGE-A4-associated cancer. (Item 43) The MAGE-A4-associated cancers are liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, metastatic solid tumor, colon cancer, gastric cancer 44. The method of claim 43, wherein the cancer is gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, or recurrent non-small cell lung cancer. (Item 44) 44. The method of any one of items 41 to 43, wherein the plurality of cells is administered to the subject in combination with a second therapeutic agent. (Item 45) 1. A T cell receptor (TCR) that specifically binds to the HLA-A2-presented cancer-testis antigen melanoma associated antigen 4 (MAGE-A4) peptide, comprising the amino acid sequence of GVYDGREHTV (SEQ ID NO: 612) (MAGE-A4 230-239), wherein the TCR comprises a complementarity-determining region 3 (CDR3) comprised in conjunction with an alpha chain variable domain of any one of SEQ ID NOs: 620, 636, 652, 668, 684, 700, 716, 732, 748, 764, 780, 796, 812, 828, 844, and 860. (Item 46) A T cell receptor (TCR) that specifically binds to the HLA-A2-presented cancer-testis antigen melanoma-associated antigen 4 (MAGE-A4) peptide, comprising the amino acid sequence of GVYDGREHTV (SEQ ID NO: 612) (MAGE-A4 230-239), and comprising a complementarity-determining region 3 (CDR3) contained within the beta chain variable domain of any one of SEQ ID NOs: 628, 644, 660, 676, 692, 708, 724, 740, 756, 772, 788, 804, 820, 836, 852, and 868. (Item 47) 46. ​​The TCR of item 45, wherein the alpha chain variable domain further comprises CDR1 and CDR2, wherein the CDR1 comprises any one of the alpha chain variable domain CDR1 amino acid sequences shown in Table 6, and the CDR2 independently comprises any one of the alpha chain variable domain CDR2 amino acid sequences shown in Table 6. (Item 48) 47. The TCR of item 46, wherein the beta chain variable domain further comprises CDR1 and CDR2, wherein the CDR1 comprises any one of the beta chain variable CDR1 amino acid sequences shown in Table 6, and the CDR2 independently comprises any one of the beta chain variable domain CDR2 amino acid sequences shown in Table 6. (Item 49) 49. The TCR of any one of items 45 to 48, wherein the TCR comprises at least one TCR alpha chain variable domain and / or at least one beta chain variable domain. (Item 50) 50. The TCR of item 49, wherein the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain. (Item 51) A TCR described in any one of items 46 to 50, comprising an alpha chain variable domain CDR1, CDR2, and CDR3 contained within any one of the alpha chain variable domain sequences listed in Table 8, and a beta chain variable domain CDR1, CDR2, and CDR3 contained within any one of the beta chain variable domain sequences listed in Table 8. (Item 52) 52. The TCR of any one of items 45 to 51, comprising an alpha chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 8. (Item 53) The TCR according to any one of Items 45 to 52, comprising a beta chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 8. (Item 54) 54. The TCR of any one of items 45 to 53, comprising: (a) an alpha chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 8; and (b) a beta chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta chain variable domain amino acid sequences listed in Table 8. (Item 55) (a) an alpha chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 614, 630, 646, 662, 678, 694, 710, 726, 742, 758, 774, 790, 806, 822, 838, and 854; (b) an alpha chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 615, 631, 647, 663, 679, 695, 711, 727, 743, 759, 775, 791, 807, 823, 839, and 855; (c) an alpha chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 616, 632, 648, 664, 680, 696, 712, 728, 744, 760, 776, 792, 808, 824, 840, and 856; (d) a beta chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 622, 638, 654, 670, 686, 702, 718, 734, 750, 766, 782, 798, 814, 830, 846, and 862; (e) a beta chain variable domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 623, 639, 655, 671, 687, 703, 719, 735, 751, 767, 783, 799, 815, 831, 847, and 863; (f) a beta chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 624, 640, 656, 672, 688, 704, 720, 736, 752, 768, 784, 800, 816, 832, 848, and 864. (Item 56) 56. The TCR of item 55, comprising an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of SEQ ID NOs: 620 / 628, 636 / 644, 652 / 660, 668 / 676, 684 / 692, 700 / 708, 716 / 724, 732 / 740, 748 / 756, 764 / 772, 780 / 788, 796 / 804, 812 / 820, 828 / 836, 844 / 852, and 860 / 868. (Item 57) SEQ ID NOs: 620 / 628, 620 / 644, 620 / 660, 620 / 676, 620 / 692, 620 / 708, 620 / 724, 620 / 740, 620 / 756, 620 / 772, 620 / 788, 620 / 804, 620 / 820, 620 / 836, 620 / 852, 620 / 868, 636 / 628, 636 / 644, 636 / 660, 636 / 676, 636 / 692, 636 / 708, 636 / 724, 636 / 740, 636 / 756, 636 / 772, 636 / 788, 636 / 804, 636 / 820, 636 / 836, 636 / 8 52, 636 / 868, 652 / 628, 652 / 644, 652 / 660, 652 / 676, 652 / 692, 652 / 708, 652 / 724, 652 / 740, 652 / 756, 652 / 772, 652 / 788, 652 / 804, 652 / 820, 652 / 836, 6 52 / 852, 652 / 868, 668 / 628, 668 / 644, 668 / 660, 668 / 676, 668 / 692, 668 / 708, 668 / 724, 668 / 740, 668 / 756, 668 / 772, 668 / 788, 668 / 804, 668 / 820, 668 / 83 6, 668 / 852, 668 / 868, 684 / 628, 684 / 644, 684 / 660, 684 / 676, 684 / 692, 684 / 708, 684 / 724, 684 / 740, 684 / 756, 684 / 772, 684 / 788, 684 / 804, 684 / 820, 68 4 / 836, 684 / 852, 684 / 868, 700 / 628, 700 / 644, 700 / 660, 700 / 676, 700 / 692, 700 / 708, 700 / 724, 700 / 740, 700 / 756, 700 / 772, 700 / 788, 700 / 804, 700 / 820 , 700 / 836, 700 / 852, 700 / 868, 716 / 628, 716 / 644, 716 / 660, 716 / 676, 716 / 692, 716 / 708, 716 / 724, 716 / 740, 716 / 756, 716 / 772, 716 / 788, 716 / 804, 716 / 820, 716 / 836, 716 / 852, 716 / 868, 732 / 628, 732 / 644, 732 / 660, 732 / 676, 732 / 692, 732 / 708, 732 / 724, 732 / 740, 732 / 756, 732 / 772, 732 / 788, 732 / 804,732 / 820、732 / 836、732 / 852、732 / 868、748 / 628、748 / 644、748 / 660、748 / 676、748 / 692、748 / 708、748 / 724、748 / 740、748 / 756、748 / 772、748 / 788、748 / 804、748 / 820、748 / 836、748 / 852、748 / 868、764 / 628、764 / 644、764 / 660、764 / 676、764 / 692、764 / 708、764 / 724、764 / 740、764 / 756、764 / 772、764 / 788、764 / 804、764 / 820、764 / 836、764 / 852、764 / 868、780 / 628、780 / 644、780 / 660、780 / 676、780 / 692、780 / 708、780 / 724、780 / 740、780 / 756、780 / 772、780 / 788、780 / 804、780 / 820、780 / 836、780 / 852、780 / 868、796 / 628、796 / 644、796 / 660、796 / 676、796 / 692、796 / 708、796 / 724、796 / 740、796 / 756、796 / 772、796 / 788、796 / 804、796 / 820、796 / 836、796 / 852、796 / 868、812 / 628、812 / 644、812 / 660、812 / 676、812 / 692、812 / 708、812 / 724、812 / 740、812 / 756、812 / 772、812 / 788、812 / 804、812 / 820、812 / 836、812 / 852、812 / 868、828 / 628、828 / 644、828 / 660、828 / 676、828 / 692、828 / 708、828 / 724、828 / 740、828 / 756、828 / 772、828 / 788、828 / 804、828 / 820、828 / 836、828 / 852、828 / 868、844 / 628、844 / 644、844 / 660、844 / 676、844 / 692、844 / 708、844 / 724、844 / 740、844 / 756、844 / 772、844 / 788、844 / 804、844 / 820、844 / 836、844 / 852、844 / 868、860 / 628、860 / 644、860 / 660、860 / 676、860 / 692、860 / 708、860 / 724、860 / 740、860 / 756、56. The TCR of item 55, comprising an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of 860 / 772, 860 / 788, 860 / 804, 860 / 820, 860 / 836, 860 / 852, and 860 / 868. (Item 58) 58. The TCR of any one of items 45 to 57, further comprising a detectable moiety. (Item 59) 58. The TCR of any one of items 45 to 57, wherein the isolated TCR has an on-target binding / off-target binding value of more than 5, more than 10, more than 15, more than 20, more than 50, more than 100, more than 200, more than 300, more than 400, more than 500, more than 600, more than 700, more than 800, more than 900, or more than 1000. (Item 60) 62. The TCR of item 61, wherein the isolated TCR has an on-target binding / off-target binding value of greater than 10. (Item 61) 62. The TCR of item 61, wherein the isolated TCR has an on-target binding / off-target binding value of greater than 500. (Item 62) A TCR that competes for binding with the isolated TCR of any one of items 45 to 61. (Item 63) 62. A pharmaceutical composition comprising the TCR according to any one of items 45 to 61 and a pharmaceutically acceptable carrier or diluent. (Item 64) An isolated cell presenting the TCR according to any one of items 45 to 61. (Item 65) A polynucleotide molecule comprising a polynucleotide sequence encoding the alpha chain variable domain of the TCR according to any one of items 45, 47, and 49 to 61. (Item 66) 62. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the beta chain variable domain of the TCR according to any one of items 46, 48, and 49 to 61. (Item 67) 67. A vector comprising the polynucleotide sequence of item 65 or 66. (Item 68) 68. An isolated cell expressing the vector of item 67. (Item 69) 63. A method for treating a subject having a MAGE-A4-related disease or disorder, comprising administering to the subject a therapeutically effective amount of the TCR of any one of items 45 to 61, the pharmaceutical composition of item 63, or the isolated cell of item 64, thereby treating the subject. (Item 70) 70. The method of claim 69, wherein the MAGE-A4 associated disease or disorder is a MAGE-A4 associated cancer. (Item 71) The MAGE-A4-associated cancers are liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, metastatic solid tumor, colon cancer, gastric cancer 71. The method of claim 70, wherein the cancer is gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, or recurrent non-small cell lung cancer. (Item 72) 72. The method of any one of items 69 to 71, wherein the TCR, the pharmaceutical composition, or the cells are administered to the subject in combination with a second therapeutic agent. (Item 73) The method according to any one of Items 69 to 71, wherein the administration is parenteral. (Item 74) 1. A polynucleotide molecule encoding a T cell receptor (TCR), wherein the TCR specifically binds to an HLA-A2-presented cancer-testis antigen melanoma associated antigen 4 (MAGE-A4) peptide comprising the amino acid sequence of GVYDGREHTV (SEQ ID NO: 612) (MAGE-A4 230-239), wherein the TCR has a property selected from the group consisting of: (a) not binding to cells expressing predicted off-target peptides, as determined by a luminescence assay; (b) not binding to cells expressing predicted off-target peptides, as determined by a flow cytometry assay; (c) activating T cell responses to approximately two-fold greater than patient-derived MAGE-A4-specific TCRs, as determined by a TCR-mediated T cell signaling luminescence bioassay; and (d) activating T cell responses to approximately two-fold greater than affinity-matured (e.g., by phage display) MAGE-A4-specific TCRs, as determined by a TCR-mediated T cell signaling luminescence bioassay. (Item 75) 75. The polynucleotide molecule of item 74, encoding at least one TCR alpha chain variable domain and / or at least one beta chain variable domain. (Item 76) 75. The polynucleotide molecule of item 74, wherein the TCR comprises an alpha chain variable domain complementarity determining region (CDR) 1, CDR2, and CDR3 comprised within any one of the alpha chain variable domain sequences listed in Table 8, and a beta chain variable domain CDR1, CDR2, and CDR3 comprised within any one of the beta chain variable domain sequences listed in Table 8. (Item 77) 77. The polynucleotide molecule of item 75 or 76, wherein the TCR comprises an alpha chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 8. (Item 78) 78. The polynucleotide molecule of any one of Items 74 to 77, wherein the TCR comprises a beta chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 8. (Item 79) 79. The polynucleotide molecule of any one of Items 74 to 78, wherein the TCR comprises: (a) an alpha chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the alpha chain variable domain amino acid sequences listed in Table 8; and (b) a beta chain variable domain having an amino acid sequence that has at least 85% amino acid identity to the entire amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 8. (Item 80) The TCR (a) an alpha chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 614, 630, 646, 662, 678, 694, 710, 726, 742, 758, 774, 790, 806, 822, 838, and 854; (b) an alpha chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 615, 631, 647, 663, 679, 695, 711, 727, 743, 759, 775, 791, 807, 823, 839, and 855; (c) an alpha chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 616, 632, 648, 664, 680, 696, 712, 728, 744, 760, 776, 792, 808, 824, 840, and 856; (d) a beta chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 622, 638, 654, 670, 686, 702, 718, 734, 750, 766, 782, 798, 814, 830, 846, and 862; (e) a beta chain variable domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 623, 639, 655, 671, 687, 703, 719, 735, 751, 767, 783, 799, 815, 831, 847, and 863; (f) a beta chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 624, 640, 656, 672, 688, 704, 720, 736, 752, 768, 784, 800, 816, 832, 848, and 864. (Item 81) 81. The polynucleotide molecule of item 80, wherein the TCR comprises an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of SEQ ID NOs: 620 / 628, 636 / 644, 652 / 660, 668 / 676, 684 / 692, 700 / 708, 716 / 724, 732 / 740, 748 / 756, 764 / 772, 780 / 788, 796 / 804, 812 / 820, 828 / 836, 844 / 852, and 860 / 868. (Item 82) The TCR is selected from the group consisting of SEQ ID NOs: 620 / 628, 620 / 644, 620 / 660, 620 / 676, 620 / 692, 620 / 708, 620 / 724, 620 / 740, 620 / 756, 620 / 772, 620 / 788, 620 / 804, 620 / 820, 620 / 836, 620 / 852, 620 / 868, 636 / 628, 636 / 644, 636 / 660, 636 / 676, 636 / 692, 636 / 708, 636 / 724, 636 / 740, 636 / 756, 636 / 772, 636 / 788, 636 / 804, 636 / 820, 636 / 8 36, 636 / 852, 636 / 868, 652 / 628, 652 / 644, 652 / 660, 652 / 676, 652 / 692, 652 / 708, 652 / 724, 652 / 740, 652 / 756, 652 / 772, 652 / 788, 652 / 804, 652 / 820, 6 52 / 836, 652 / 852, 652 / 868, 668 / 628, 668 / 644, 668 / 660, 668 / 676, 668 / 692, 668 / 708, 668 / 724, 668 / 740, 668 / 756, 668 / 772, 668 / 788, 668 / 804, 668 / 82 0, 668 / 836, 668 / 852, 668 / 868, 684 / 628, 684 / 644, 684 / 660, 684 / 676, 684 / 692, 684 / 708, 684 / 724, 684 / 740, 684 / 756, 684 / 772, 684 / 788, 684 / 804, 68 4 / 820, 684 / 836, 684 / 852, 684 / 868, 700 / 628, 700 / 644, 700 / 660, 700 / 676, 700 / 692, 700 / 708, 700 / 724, 700 / 740, 700 / 756, 700 / 772, 700 / 788, 700 / 804 , 700 / 820, 700 / 836, 700 / 852, 700 / 868, 716 / 628, 716 / 644, 716 / 660, 716 / 676, 716 / 692, 716 / 708, 716 / 724, 716 / 740, 716 / 756, 716 / 772, 716 / 788, 716 / 804, 716 / 820, 716 / 836, 716 / 852, 716 / 868, 732 / 628, 732 / 644, 732 / 660, 732 / 676, 732 / 692, 732 / 708, 732 / 724, 732 / 740, 732 / 756, 732 / 772, 732 / 788,732 / 804、732 / 820、732 / 836、732 / 852、732 / 868、748 / 628、748 / 644、748 / 660、748 / 676、748 / 692、748 / 708、748 / 724、748 / 740、748 / 756、748 / 772、748 / 788、748 / 804、748 / 820、748 / 836、748 / 852、748 / 868、764 / 628、764 / 644、764 / 660、764 / 676、764 / 692、764 / 708、764 / 724、764 / 740、764 / 756、764 / 772、764 / 788、764 / 804、764 / 820、764 / 836、764 / 852、764 / 868、780 / 628、780 / 644、780 / 660、780 / 676、780 / 692、780 / 708、780 / 724、780 / 740、780 / 756、780 / 772、780 / 788、780 / 804、780 / 820、780 / 836、780 / 852、780 / 868、796 / 628、796 / 644、796 / 660、796 / 676、796 / 692、796 / 708、796 / 724、796 / 740、796 / 756、796 / 772、796 / 788、796 / 804、796 / 820、796 / 836、796 / 852、796 / 868、812 / 628、812 / 644、812 / 660、812 / 676、812 / 692、812 / 708、812 / 724、812 / 740、812 / 756、812 / 772、812 / 788、812 / 804、812 / 820、812 / 836、812 / 852、812 / 868、828 / 628、828 / 644、828 / 660、828 / 676、828 / 692、828 / 708、828 / 724、828 / 740、828 / 756、828 / 772、828 / 788、828 / 804、828 / 820、828 / 836、828 / 852、828 / 868、844 / 628、844 / 644、844 / 660、844 / 676、844 / 692、844 / 708、844 / 724、844 / 740、844 / 756、844 / 772、844 / 788、844 / 804、844 / 820、844 / 836、844 / 852、844 / 868、860 / 628、860 / 644、860 / 660、860 / 676、860 / 692、860 / 708、860 / 724、860 / 740、81. The polynucleotide molecule of item 80, comprising an alpha chain variable domain / beta chain variable domain amino acid sequence pair selected from the group consisting of 860 / 756, 860 / 772, 860 / 788, 860 / 804, 860 / 820, 860 / 836, 860 / 852, and 860 / 868. (Item 83) The TCR (a) an alpha chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 617, 633, 649, 665, 681, 697, 713, 729, 745, 761, 777, 793, 809, 825, 841, and 857; (b) an alpha chain variable domain CDR2 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 618, 634, 650, 666, 682, 698, 714, 730, 746, 762, 778, 794, 810, 826, 842, and 858; (c) an alpha chain variable domain CDR3 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 619, 635, 651, 667, 683, 699, 715, 731, 747, 763, 779, 795, 811, 827, 843, and 859; (d) a beta chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 625, 641, 657, 673, 689, 705, 721, 737, 753, 769, 785, 801, 817, 833, 849, and 865; (e) a beta chain variable domain CDR2 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 626, 642, 658, 674, 690, 706, 722, 738, 754, 770, 786, 802, 818, 834, 850, and 866; (f) a beta chain variable domain CDR3 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 627, 643, 659, 675, 691, 707, 723, 739, 755, 771, 787, 803, 819, 835, 851, and 867. (Item 84) 84. The isolated nucleic acid molecule of claim 83, wherein the TCR comprises an alpha chain variable domain / beta chain variable domain nucleic acid sequence pair selected from the group consisting of SEQ ID NOs: 621 / 629, 637 / 645, 653 / 661, 669 / 677, 685 / 693, 701 / 709, 717 / 725, 733 / 741, 749 / 757, 765 / 773, 781 / 789, 797 / 805, 813 / 821, 829 / 837, 845 / 853, and 861 / 869. (Item 85) A vector comprising the polynucleotide sequence of the isolated nucleic acid molecule according to any one of Items 74 to 84. (Item 86) 86. An isolated cell comprising the vector of item 85. (Item 87) 87. A method of treating a subject having a MAGE-A4-related disease or disorder, comprising administering to the subject the cells of item 86, thereby treating the subject. (Item 88) 88. The method of claim 87, wherein the MAGE-A4 associated disease or disorder is a MAGE-A4 associated cancer. (Item 89) The MAGE-A4-associated cancers are liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, metastatic solid tumor, colon cancer, gastric cancer 89. The method of claim 88, wherein the cancer is gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, or recurrent non-small cell lung cancer. (Item 90) 90. The method of any one of items 87 to 89, wherein the cells are administered to the subject in combination with a second therapeutic agent.

Claims

[Claim 1] The invention described in the present specification.

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