(PRAME) T-cell receptors that are preferentially expressed in melanoma and methods of using them.

Abstract

To provide (PRAME) T-cell receptors that are preferentially expressed in melanoma, and methods for using them. [Solution] The present invention provides isolated T cell receptors (TCRs) that specifically bind to the PRAME peptide, an HLA-presenting cancer testicular antigen preferentially expressed in melanoma, as well as therapeutic and diagnostic methods using these isolated TCRs. The present invention provides T cell receptors (TCRs) created for the PRAME peptide antigen in association with MHC (HLA-A2). The identified unique TCR sequences show specific binding to the small peptide PRAME present in the groove of the HLA molecule.

Description

[Technical Field] 【0001】 Related applications This application claims priority to U.S. Provisional Application No. 62 / 965,231, filed on 24 January 2020, the entirety of which is incorporated herein by reference. 【0002】 Sequence List This application includes an array listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on January 20, 2021, is named 118003-00520_SL.txt and has a size of 461,085 bytes. [Background technology] 【0003】 The T cell receptor (TCR) is a membrane-bound heterodimer containing α and β chains similar to the immunoglobulin variable (V) and constant (C) regions. The TCR α chain contains covalently linked V-α and C-α chains, while the β chain contains a V-β chain covalently linked to the C-β chain. The V-α and V-β chains form a pocket or gap that can bind to antigens in association with the major histocompatibility complex (MHC) (known as the HLA complex in humans). (Davis Ann. Rev. of Immunology 3:537 (1985); Fundamental Immunology 3rd Ed., W. Paul Ed. New York (1993)). 【0004】 TCRs are key effectors of the immune system, possessing unique advantages as a platform for developing therapeutic agents. While antibody therapies are limited to the recognition of 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 subtype of T cell that recognizes and is activated by the presented antigen, TCRs can be involved in regulating various immune responses. For example, T cells are involved in regulating humoral immune responses by inducing the differentiation of B cells into antibody-producing cells. Furthermore, activated T cells act to initiate cell-mediated immune responses. Thus, TCRs can recognize additional targets that are not available to antibodies. In addition, TCRs have been reported to mediate cell death, increase B cell proliferation, and influence the onset 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 due to their ability to redirect T cells to tumors expressing antigens. TCRs bind to small peptides consisting of only 8-12 amino acids in length, which are bound to the surface of target cells by the major histocompatibility complex (MHC). Thus, TCRs can recognize intracellular antigens or viral proteins derived from cancer because these antigens are processed and presented as peptides in association with surface MHC. Therefore, TCRs can recognize additional internal cellular targets that are not available to antibodies or therapies that cannot penetrate cells. 【0006】 However, the challenge in this industry is to manipulate TCRs that, when administered to patients, lack immunogenicity and possess fine specificity for specific peptide antigens of interest, without cross-reacting with similar epitopes found in other peptides on the MHC or in the natural protein repertoire. 【0007】 PRAME, an antigen preferentially expressed in melanoma, is a well-known cancer-testis antigen (CTA) encoded on the X chromosome. It was first identified as a tumor antigen recognizable by HLA-A*24-limited cytotoxic T lymphocytes in metastatic cutaneous melanoma (Ikeda H., et al. (1997) Immunity 6:199-208). PRAME has been shown to function as a repressor of the retinoic acid receptor, and therefore may confer a proliferative advantage to cancer cells through this mechanism (see, for example, Epping MT, et al. (2005) Cell 122:835-847). 【0008】 PRAME is richly reexpressed in many tumors of different histological types, including melanoma, renal cell carcinoma, non-small cell lung cancer (NSCLC), neuroblastoma, breast cancer, multiple myeloma, acute leukemia, chronic myeloid leukemia, multiple sarcoma subtypes, and primary and metastatic uveal melanoma, but in normal, healthy adult tissue, PRAME expression is limited to the testes. There is an unmet need in the art for novel targeted agents based on T cell receptors that specifically bind to PRAME antigens, as well as for methods for manufacturing and using such agents in therapeutic and diagnostic settings. [Prior art documents] [Non-patent literature] 【0009】 [Non-Patent Document 1] Davis Ann.Rev.of Immunology 3:537(1985);Fundamental Immunology 3rd Ed., W.Paul Ed.New York(1993). [Non-Patent Document 2] Ikeda H., et al. (1997) Immunity 6:199-208 [Non-Patent Document 3] Epping MT, et al. (2005) Cell 122:835-847 [Overview of the Initiative] [Means for solving the problem] 【0010】 This invention provides a T cell receptor (TCR) created for the PRAME peptide antigen in association with MHC (HLA-A2). The identified unique TCR sequence exhibits specific binding to the small peptide PRAME present in the groove of the HLA molecule. In embodiments of the present invention, for example, the following items are provided. (Item 1) A T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, comprising the amino acid sequence RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), wherein the TCR comprises an alpha-chain variable domain including a complementary determinant region (CDR) 3, and the CDR 3 comprises one of the amino acid sequences of the alpha-chain variable domain CDR3 shown in Table 3. (Item 2) A T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, comprising the amino acid sequence RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), wherein the TCR comprises a beta-chain variable domain including a complementary determinant region (CDR)3, and the CDR3 comprises any one of the beta-chain variable domain CDR3 amino acid sequences shown in Table 3. (Item 3) The TCR according to item 1 or 2, wherein the TCR comprises at least one TCR alpha-chain variable domain and / or at least one beta-chain variable domain. (Item 4) The TCR described in item 3, wherein the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain. (Item 5) The TCR according to any one of items 1 to 4, wherein the alpha-chain variable domain further comprises CDR1 and CDR2, wherein CDR1 comprises one of the alpha-chain variable domain CDR1 amino acid sequences shown in Table 3, and CDR2 independently comprises one of the alpha-chain variable domain CDR2 amino acid sequences shown in Table 3. (Item 6) The TCR according to item 5, wherein 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 3, and CDR2 independently comprises any one of the beta-chain variable domain CDR2 amino acid sequences shown in Table 3. (Item 7) A TCR as described in any one of items 4 to 6, comprising alpha-chain variable domains CDR1, CDR2, and CDR3 contained within any one of the alpha-chain variable domain sequences listed in Table 5, and beta-chain variable domains CDR1, CDR2, and CDR3 contained within any one of the beta-chain variable domain sequences listed in Table 5. (Item 8) RLDQLLRHV (Sequence code 929) (PRAME A T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, comprising the amino acid sequence of 312-320), wherein the TCR comprises alpha chain CDR1, CDR2, and CDR3 / beta chain CDR1, CDR2, and CDR3 amino acid sequences selected from the alpha chain / beta chain variable domain sequence pairs of SEQ ID NOs: 217 / 219, 221 / 223, 225 / 227, 229 / 231, 233 / 235, 237 / 239, 241 / 243, 245 / 247, 249 / 251, 253 / 255, 257 / 259, 261 / 263, 265 / 267, 269 / 271, 273 / 275, 277 / 279, 281 / 283, and 285 / 287. (Item 9) A TCR according to any one of items 1 to 8, comprising an alpha-chain variable domain having an amino acid sequence with 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 5. (Item 10) A TCR according to any one of items 1 to 9, comprising a beta-chain variable domain having an amino acid sequence with 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 5. (Item 11) (a) An alpha-chain variable domain having an amino acid sequence with 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 5, and (b) a beta-chain variable domain having an amino acid sequence with 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 5, a TCR according to any one of items 1 to 10. (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, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, and 103, (b) An alpha-chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, and 104, (c) An alpha-chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, and 105, (d) A beta-chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, and 106, (e) A beta-chain variable domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, and 107, (f) A TCR according to any one of items 4 to 11, comprising a beta-chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, and 108. (Item 13) The TCR described in item 12, comprising an alpha-chain variable domain / beta-chain variable domain amino acid sequence pair selected from the group consisting of SEQ ID NOs: 217 / 219, 221 / 223, 225 / 227, 229 / 231, 233 / 235, 237 / 239, 241 / 243, 245 / 247, 249 / 251, 253 / 255, 257 / 259, 261 / 263, 265 / 267, 269 / 271, 273 / 275, 277 / 279, 281 / 283, and 285 / 287. (Item 14) A T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, comprising the amino acid sequence SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), wherein the TCR comprises an alpha-chain variable domain including a complementary determinant region (CDR) 3, and the CDR 3 comprises one of the amino acid sequences of the alpha-chain variable domain CDR3 shown in Table 6. (Item 15) A T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, comprising the amino acid sequence SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), wherein the TCR comprises a beta-chain variable domain including a complementary determinant region (CDR) 3, and the CDR3 comprises one of the amino acid sequences of the beta-chain variable domain CDR3 shown in Table 6. (Item 16) The TCR according to item 14 or 15, wherein 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. (Item 17) The TCR according to item 16, wherein 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. (Item 18) The TCR described in any one of items 14 to 17, wherein the TCR comprises at least one TCR alpha-chain variable domain and / or at least one beta-chain variable domain. (Item 19) The TCR described in item 18, wherein the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain. (Item 20) A TCR as described in any one of items 17 to 19, comprising alpha-chain variable domains CDR1, CDR2, and CDR3 contained within any one of the alpha-chain variable domain sequences listed in Table 8, and beta-chain variable domains CDR1, CDR2, and CDR3 contained within any one of the beta-chain variable domain sequences listed in Table 8. (Item 21) A T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, comprising the amino acid sequence SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), wherein the TCR is associated with SEQ ID NOs: 769 / 771, 773 / 775, 777 / 779, 781 / 783, 785 / 787, 789 / 791, 793 / 795, 797 / 799, 801 / 803, 805 / 807, 809 / 811, 813 / 815, 817 / 819, 821 / 823, 825 / 827, 829 / 831, 833 / 835, 837 / 839, 841 / 843, 845 / 8 TCRs comprising alpha-chain CDR1, CDR2, and CDR3 / beta-chain CDR1, CDR2, and CDR3 amino acid sequences selected from alpha-chain / beta-chain variable domain sequence pairs of 47, 849 / 851, 853 / 855, 857 / 859, 861 / 863, 865 / 867, 869 / 871, 873 / 875, 877 / 879, 881 / 883, 885 / 887, 889 / 891, 893 / 805, 897 / 899, 901 / 903, 905 / 907, 909 / 911, 913 / 915, 917 / 919, 921 / 923, and 925 / 927. (Item 22) A TCR according to any one of items 14 to 21, comprising an alpha-chain variable domain having an amino acid sequence that has at least 85% amino acid identity with respect to the entire amino acid sequence of any one of the alpha-chain variable domain amino acid sequences listed in Table 8. (Item 23) A TCR according to any one of items 14 to 22, comprising a beta-chain variable domain having an amino acid sequence that has at least 85% amino acid identity with respect to the entire amino acid sequence of any one of the beta-chain variable domain amino acid sequences listed in Table 8. (Item 24) A TCR according to any one of items 14 to 23, comprising: (a) an alpha-chain variable domain having an amino acid sequence having at least 85% amino acid identity with respect to any one of the amino acid sequences listed in Table 8; and (b) a beta-chain variable domain having an amino acid sequence having at least 85% amino acid identity with respect to any one of the amino acid sequences listed in Table 8. (Item 25) (a) an alpha-chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 289, 295, 301, 307, 313, 319, 325, 331, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457, 463, 469, 475, 481, 487, 493, 499, 505, 511, 517, and 523, (b) an alpha-chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 290, 296, 302, 308, 314, 320, 326, 332, 338, 344, 350, 356, 362, 368, 374, 380, 386, 392, 398, 404, 410, 416, 422, 428, 434, 440, 446, 452, 458, 464, 470, 476, 482, 488, 494, 500, 506, 512, 518, and 524, (c) Alpha-chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 291, 297, 303, 309, 315, 321, 327, 333, 339, 345, 351, 357, 363, 369, 375, 381, 387, 393, 399, 405, 411, 417, 423, 429, 435, 441, 447, 453, 459, 465, 471, 477, 483, 489, 495, 501, 507, 513, 519, and 525, (d) A beta-chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 292, 298, 304, 310, 316, 322, 328, 334, 340, 346, 352, 358, 364, 370, 376, 382, ​​388, 394, 400, 406, 412, 418, 424, 430, 436, 442, 448, 454, 460, 466, 472, 478, 484, 490, 496, 502, 508, 514, 520, and 526, (e) A beta-chain variable domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 293, 299, 305, 311, 317, 323, 329, 335, 341, 347, 353, 359, 365, 371, 377, 383, 389, 395, 401, 407, 413, 419, 425, 431, 437, 443, 449, 455, 461, 467, 473, 479, 485, 491, 497, 503, 509, 515, 521, and 527, (f) A TCR according to any one of items 17 to 24, comprising a beta-chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 294, 300, 306, 312, 318, 324, 330, 336, 342, 348, 354, 360, 366, 372, 378, 384, 390, 396, 402, 408, 414, 420, 426, 432, 438, 444, 450, 456, 462, 468, 474, 480, 486, 492, 498, 504, 510, 516, 522, and 528. (Item 26) Sequence numbers 769 / 771, 773 / 775, 777 / 779, 781 / 783, 785 / 787, 789 / 791, 793 / 795, 797 / 799, 801 / 803, 805 / 807, 809 / 811, 813 / 815, 817 / 819, 821 / 823, 825 / 827, 829 / 831, 833 / 835, 837 / 839, 841 / 843, 845 / 847, 849 / 851, 853 / 855, 857 / 859, 861 The TCR described in item 25, comprising an alpha-chain variable domain / beta-chain variable domain amino acid sequence pair selected from the group consisting of / 863, 865 / 867, 869 / 871, 873 / 875, 877 / 879, 881 / 883, 885 / 887, 889 / 891, 893 / 805, 897 / 899, 901 / 903, 905 / 907, 909 / 911, 913 / 915, 917 / 919, 921 / 923, and 925 / 927. (Item 27) A TCR that competes for binding to any of the TCRs listed in items 1 through 26. (Item 28) A TCR that binds to the same epitope as any one of the TCRs listed in items 1-26. (Item 29) The TCR described in any one of items 1 to 28, including the detectable portion. (Item 30) Isolated cells that present a TCR as described in any one of items 1-29. (Item 31) An isolated polynucleotide molecule containing a polynucleotide sequence encoding the alpha-chain variable domain of a TCR as described in any one of items 1 through 29. (Item 32) An isolated polynucleotide molecule containing a polynucleotide sequence encoding the beta-chain variable domain of a TCR as described in any one of items 1 through 29. (Item 33) A vector containing the polynucleotide molecule described in item 31 or 32. (Item 34) Isolated cells expressing the vector described in item 33. (Item 35) A pharmaceutical composition comprising isolated cells as described in item 34 and a pharmaceutically acceptable carrier or diluent. (Item 36) A method for treating a subject having a PRAME-related disease or disorder, comprising administering to the subject a therapeutically effective amount of a TCR described in any one of items 1 to 29, a pharmaceutical composition described in item 35, or a plurality of isolated cells described in item 30, thereby treating the subject. (Item 37) The method according to item 36, wherein the PRAME-related disease or disorder is PRAME-related cancer. (Item 38) The aforementioned PRAME-related 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 carcinoma, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, undifferentiated astrocytoma, brain tumor, fallopian tube cancer, primary abdominal 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, colorectal cancer, stomach cancer, gastric cancer The method described in item 37, which is cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, endometrial cancer, uterine carcinosarcoma, testicular germ cell tumor, uveal melanoma, renal papillary cell carcinoma, renal clear cell carcinoma, thymoma, colon adenocarcinoma, cervical squamous cell carcinoma, cervical tumor, pancreatic adenocarcinoma, liver cancer, hepatocellular carcinoma, mesothelioma, or recurrent non-small cell lung cancer. (Item 39) The method according to any one of items 36 to 38, wherein the TCR, the pharmaceutical composition, or the plurality of cells are administered to the subject in combination with a second therapeutic agent. (Item 40) The method according to any one of items 36 to 39, wherein the TCR, the pharmaceutical composition, or the plurality of cells are administered to the subject subcutaneously, intravenously, intradermally, intraperitoneally, or orally, intramuscularly, or intracranially. (Item 41) A polynucleotide molecule encoding a T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, comprising the amino acid sequence RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), wherein the TCR comprises an alpha-chain variable domain including a complementary determinant region (CDR) 3, and the CDR 3 comprises one of the amino acid sequences of the alpha-chain variable domain CDR3 shown in Table 5. (Item 42) A polynucleotide molecule encoding a T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, comprising the amino acid sequence RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), wherein the TCR comprises a beta-chain variable domain including a complementary determinant region (CDR)3, and the CDR3 comprises one of the amino acid sequences of the beta-chain variable domain CDR3 shown in Table 5. (Item 43) A polynucleotide molecule as described in item 41 or 42, encoding at least one TCR alpha-chain variable domain and / or at least one beta-chain variable domain. (Item 44) The polynucleotide molecule described in item 43, wherein the TCR comprises alpha-chain variable domain complementary determination regions (CDRs) 1, 2, and 3 contained within any one of the alpha-chain variable domain sequences listed in Table 5, and beta-chain variable domains CDR1, 2, and 3 contained within any one of the beta-chain variable domain sequences listed in Table 5. (Item 45) A polynucleotide molecule encoding a T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, comprising the amino acid sequence RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), wherein the TCR is a T-cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, and the TCR is a T-cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide that contains the amino acid sequence RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), and the TCR is a T-cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide that contains the amino acid sequence RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), and the TCR is a T-cell receptor (TCR) that contains the amino acid sequence RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), and the TCR is a T-cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide that preferentially binds to an antigen (PRAME), and the T-cell receptor (TCR) is a T-cell receptor (TCR) that contains the amino acid sequence RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), and the T-cell receptor (TCR) A polynucleotide molecule comprising alpha-chain CDR1, CDR2, and CDR3 / beta-chain CDR1, CDR2, and CDR3 amino acid sequences selected from alpha-chain / beta-chain variable domain sequence pairs of 49 / 251, 253 / 255, 257 / 259, 261 / 263, 265 / 267, 269 / 271, 273 / 275, 277 / 279, 281 / 283, and 285 / 287. (Item 46) A polynucleotide molecule according to any one of items 41 to 45, wherein the TCR comprises an alpha-chain variable domain having an amino acid sequence that has at least 85% amino acid identity with respect to the entire amino acid sequence of any one of the alpha-chain variable domain amino acid sequences listed in Table 5. (Item 47) A polynucleotide molecule according to any one of items 41 to 46, wherein the TCR comprises a beta-chain variable domain having an amino acid sequence that has at least 85% amino acid identity with respect to the entire amino acid sequence of any one of the beta-chain variable domain amino acid sequences listed in Table 5. (Item 48) A polynucleotide molecule according to any one of items 41 to 47, wherein the TCR comprises (a) an alpha-chain variable domain having an amino acid sequence having at least 85% amino acid identity with respect to the entire amino acid sequence of any one of the alpha-chain variable domain amino acid sequences listed in Table 5, and (b) a beta-chain variable domain having an amino acid sequence having at least 85% amino acid identity with respect to the entire amino acid sequence of any one of the beta-chain variable domain amino acid sequences listed in Table 5. (Item 49) The TCR comprises an alpha-chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, and 103, (b) an alpha-chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, and 104, (c) Alpha chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, and 105, (d) A beta-chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, and 106, (e) A beta-chain variable domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, and 107, (f) A polynucleotide molecule according to any one of items 41 to 48, comprising a beta-chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, and 108. (Item 50) The polynucleotide molecule described in item 48, 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: 217 / 219, 221 / 223, 225 / 227, 229 / 231, 233 / 235, 237 / 239, 241 / 243, 245 / 247, 249 / 251, 253 / 255, 257 / 259, 261 / 263, 265 / 267, 269 / 271, 273 / 275, 277 / 279, 281 / 283, and 285 / 287. (Item 51) 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: 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, and 211, (b) Alpha-chain variable domain CDR2 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, and 212, (c) Alpha-chain variable domain CDR3 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, and 213, (d) A beta-chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, and 214, (e) A beta-chain variable domain CDR2 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, and 215, (f) A polynucleotide molecule as described in item 48, comprising a beta-chain variable domain CDR3 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, and 216. (Item 52) The polynucleotide molecule according to item 51, 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: 218 / 220, 222 / 224, 226 / 228, 230 / 232, 234 / 236, 238 / 240, 242 / 244, 246 / 248, 250 / 252, 254 / 256, 258 / 260, 262 / 264, 266 / 268, 270 / 272, 274 / 276, 278 / 280, 282 / 284, and 286 / 288. (Item 53) A vector containing a polynucleotide molecule as described in any one of items 41-52. (Item 54) Isolated cells containing the vector described in item 53. (Item 55) A polynucleotide molecule encoding a T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, comprising the amino acid sequence SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), wherein the TCR comprises an alpha-chain variable domain including a complementary determinant region (CDR)3, and the CDR3 comprises one of the amino acid sequences of the alpha-chain variable domain CDR3 shown in Table 8. (Item 56) A polynucleotide molecule encoding a T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, comprising the amino acid sequence SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), wherein the TCR comprises a beta-chain variable domain including a complementary determinant region (CDR)3, and the CDR3 comprises one of the amino acid sequences of the beta-chain variable domain CDR3 shown in Table 8. (Item 57) A polynucleotide molecule as described in item 55 or 56, encoding at least one TCR alpha-chain variable domain and / or at least one beta-chain variable domain. (Item 58) A polynucleotide molecule encoding a T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, containing the amino acid sequence SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), wherein the TCR is associated with SEQ ID NOs: 769 / 771, 773 / 775, 777 / 779, 781 / 783, 785 / 787, 789 / 791, 793 / 795, 797 / 799, 801 / 803, 805 / 807, 809 / 811, 813 / 815, 817 / 819, 821 / 823, 825 / 827, 829 / 831, 833 / 835, 837 / 839, 841 / 843, 84 A polynucleotide molecule comprising alpha-chain CDR1, CDR2, and CDR3 / beta-chain CDR1, CDR2, and CDR3 amino acid sequences selected from alpha-chain / beta-chain variable domain sequence pairs of 5 / 847, 849 / 851, 853 / 855, 857 / 859, 861 / 863, 865 / 871, 873 / 875, 877 / 879, 881 / 883, 885 / 887, 889 / 891, 893 / 805, 897 / 899, 901 / 903, 905 / 907, 909 / 911, 913 / 915, 917 / 919, 921 / 923, and 925 / 927. (Item 59) The polynucleotide molecule described in item 58, wherein the TCR comprises alpha-chain variable domain complementary determination regions (CDRs) 1, CDR2, and CDR3 contained within any one of the alpha-chain variable domain sequences listed in Table 8, and beta-chain variable domains CDR1, CDR2, and CDR3 contained within any one of the beta-chain variable domain sequences listed in Table 8. (Item 60) A polynucleotide molecule according to any one of items 55 to 59, wherein the TCR comprises an alpha-chain variable domain having an amino acid sequence that has at least 85% amino acid identity with respect to the entire amino acid sequence of any one of the alpha-chain variable domain amino acid sequences listed in Table 8. (Item 61) A polynucleotide molecule according to any one of items 55 to 60, wherein the TCR comprises a beta-chain variable domain having an amino acid sequence that has at least 85% amino acid identity with respect to the entire amino acid sequence of any one of the beta-chain variable domain amino acid sequences listed in Table 8. (Item 62) A polynucleotide molecule according to any one of items 55 to 61, wherein the TCR comprises (a) an alpha-chain variable domain having an amino acid sequence having at least 85% amino acid identity with respect 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 having at least 85% amino acid identity with respect to the entire amino acid sequence of any one of the beta-chain variable domain amino acid sequences listed in Table 8. (Item 63) The TCR comprises an alpha-chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of (a) SEQ ID NOs: 289, 295, 301, 307, 313, 319, 325, 331, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457, 463, 469, 475, 481, 487, 493, 499, 505, 511, 517, and 523, (b) an alpha-chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 290, 296, 302, 308, 314, 320, 326, 332, 338, 344, 350, 356, 362, 368, 374, 380, 386, 392, 398, 404, 410, 416, 422, 428, 434, 440, 446, 452, 458, 464, 470, 476, 482, 488, 494, 500, 506, 512, 518, and 524, (c) Alpha-chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 291, 297, 303, 309, 315, 321, 327, 333, 339, 345, 351, 357, 363, 369, 375, 381, 387, 393, 399, 405, 411, 417, 423, 429, 435, 441, 447, 453, 459, 465, 471, 477, 483, 489, 495, 501, 507, 513, 519, and 525, (d) A beta-chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 292, 298, 304, 310, 316, 322, 328, 334, 340, 346, 352, 358, 364, 370, 376, 382, ​​388, 394, 400, 406, 412, 418, 424, 430, 436, 442, 448, 454, 460, 466, 472, 478, 484, 490, 496, 502, 508, 514, 520, and 526, (e) A beta-chain variable domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 293, 299, 305, 311, 317, 323, 329, 335, 341, 347, 353, 359, 365, 371, 377, 383, 389, 395, 401, 407, 413, 419, 425, 431, 437, 443, 449, 455, 461, 467, 473, 479, 485, 491, 497, 503, 509, 515, 521, and 527, (f) A polynucleotide molecule according to any one of items 55 to 62, comprising a beta-chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 294, 300, 306, 312, 318, 324, 330, 336, 342, 348, 354, 360, 366, 372, 378, 384, 390, 396, 402, 408, 414, 420, 426, 432, 438, 444, 450, 456, 462, 468, 474, 480, 486, 492, 498, 504, 510, 516, 522, and 528. (Item 64) The aforementioned TCRs are sequence numbers 769 / 771, 773 / 775, 777 / 779, 781 / 783, 785 / 787, 789 / 791, 793 / 795, 797 / 799, 801 / 803, 805 / 807, 809 / 811, 813 / 815, 817 / 819, 821 / 823, 825 / 827, 829 / 831, 833 / 835, 837 / 839, 841 / 843, 845 / 847, 849 / 851, 853 / 855, 857 / 859, and 861. A polynucleotide molecule as described in item 63, comprising an alpha-chain variable domain / beta-chain variable domain amino acid sequence pair selected from the group consisting of / 863, 865 / 867, 869 / 871, 873 / 875, 877 / 879, 881 / 883, 885 / 887, 889 / 891, 893 / 805, 897 / 899, 901 / 903, 905 / 907, 909 / 911, 913 / 915, 917 / 919, 921 / 923, and 925 / 927. (Item 65) The TCR comprises an alpha-chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of (a) SEQ ID NOs: 529, 535, 541, 547, 553, 559, 565, 571, 577, 583, 589, 595, 601, 607, 613, 619, 625, 631, 637, 643, 649, 655, 661, 667, 673, 679, 685, 691, 697, 703, 709, 715, 721, 727, 733, 739, 745, 751, 757, and 763, (b) Alpha-chain variable domain CDR2 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 530, 536, 542, 548, 554, 560, 566, 572, 578, 584, 590, 596, 602, 608, 614, 620, 626, 632, 638, 644, 650, 656, 662, 668, 674, 680, 686, 692, 698, 704, 710, 716, 722, 728, 734, 740, 746, 752, 758, and 764, (c) Alpha-chain variable domain CDR3 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 531, 537, 543, 549, 555, 561, 567, 573, 579, 585, 591, 597, 603, 609, 615, 621, 627, 633, 639, 645, 651, 657, 663, 669, 675, 681, 687, 693, 699, 705, 711, 717, 723, 729, 735, 741, 747, 753, 759, and 765, (d) A beta-chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 532, 538, 544, 550, 556, 562, 568, 574, 580, 586, 592, 598, 604, 610, 616, 622, 628, 634, 640, 646, 652, 658, 664, 670, 676, 682, 688, 694, 700, 706, 712, 718, 724, 730, 736, 742, 748, 754, 760, and 766, (e) A beta-chain variable domain CDR2 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 533, 539, 545, 551, 557, 563, 569, 575, 581, 587, 593, 599, 605, 611, 617, 623, 629, 635, 641, 647, 653, 659, 665, 671, 677, 683, 689, 695, 701, 707, 713, 719, 725, 731, 737, 743, 749, 755, 761, and 767, (f) A polynucleotide molecule as described in item 62, comprising a beta-chain variable domain CDR3 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 534, 540, 546, 552, 558, 564, 570, 576, 582, 588, 594, 600, 606, 612, 618, 624, 630, 636, 642, 648, 654, 660, 666, 672, 678, 684, 690, 696, 702, 708, 714, 720, 726, 732, 738, 744, 750, 756, 762, and 768. (Item 66) The aforementioned TCRs are sequence numbers 770 / 772, 774 / 776, 778 / 780, 782 / 784, 786 / 788, 790 / 792, 794 / 796, 798 / 800, 802 / 804, 806 / 808, 810 / 812, 814 / 816, 818 / 820, 822 / 824, 826 / 828, 830 / 832, 834 / 836, 838 / 840, 842 / 844, 846 / 848, 850 / 852, 854 / 856, 858 / 860, 86 A polynucleotide molecule as described in item 65, comprising an alpha-chain variable domain / beta-chain variable domain nucleic acid sequence pair selected from the group consisting of 2 / 864, 866 / 868, 870 / 872, 874 / 876, 878 / 880, 882 / 884, 886 / 888, 890 / 892, 894 / 896, 898 / 900, 902 / 904, 906 / 908, 910 / 912, 914 / 916, 918 / 920, 922 / 924, and 926 / 928. (Item 67) A vector containing a polynucleotide molecule as described in any one of items 55-66. (Item 68) Isolated cells containing the vector described in item 67. (Item 69) A method for treating a subject having a PRAME-related disease or disorder, comprising administering to the subject a plurality of cells described in item 54 or 68, thereby treating the subject. (Item 70) The method according to item 69, wherein the PRAME-related disease or disorder is PRAME-related cancer. (Item 71) The aforementioned PRAME-related 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 carcinoma, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, undifferentiated astrocytoma, brain tumor, fallopian tube cancer, primary abdominal 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, colorectal cancer, stomach cancer, gastric cancer The method described in item 70, which is cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, endometrial cancer, uterine carcinosarcoma, testicular germ cell tumor, uveal melanoma, renal papillary cell carcinoma, renal clear cell carcinoma, thymoma, colon adenocarcinoma, cervical squamous cell carcinoma, cervical tumor, pancreatic adenocarcinoma, liver cancer, hepatocellular carcinoma, mesothelioma, or recurrent non-small cell lung cancer. (Item 72) The method according to any one of items 69 to 71, wherein the plurality of cells are administered to the subject in combination with a second therapeutic agent. 【0011】 Accordingly, in one embodiment, the present invention provides a T cell receptor (TCR) (e.g., an isolated TCR or a TCR expressed in isolated cells) that specifically binds to an antigen (PRAME) peptide that is preferentially expressed in melanoma and contains the amino acid sequence of RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), which is an HLA-A2 presenting cancer testicular antigen, wherein the TCR comprises an alpha chain variable domain including a complementary determinant region (CDR)3, and CDR3 comprises any one of the alpha chain variable domain CDR3 amino acid sequences shown in Table 3. 【0012】 In another embodiment, the present invention provides a T cell receptor (TCR) (e.g., an isolated TCR or a TCR expressed in isolated cells) that specifically binds to an antigen (PRAME) peptide that is preferentially expressed in melanoma and contains the amino acid sequence of RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), which is an HLA-A2 presenting cancer testicular antigen, wherein the TCR comprises a beta-chain variable domain including a complementary determinant region (CDR)3, and CDR3 comprises any one of the beta-chain variable domain CDR3 amino acid sequences shown in Table 3. 【0013】 In some embodiments, the alpha-chain variable domain further comprises CDR1 and CDR2, where CDR1 comprises one of the alpha-chain variable domain CDR1 amino acid sequences shown in Table 3, and CDR2 independently comprises one of the alpha-chain variable domain CDR2 amino acid sequences shown in Table 3. 【0014】 In some embodiments, the beta-chain variable domain further comprises CDR1 and CDR2, where CDR1 comprises any one of the beta-chain variable CDR1 amino acid sequences shown in Table 3, and CDR2 independently comprises any one of the beta-chain variable domain CDR2 amino acid sequences shown in Table 3. 【0015】 The TCR may include at least one TCR alpha-chain variable domain and / or at least one TCR beta-chain variable domain, or the TCR may include both a TCR alpha-chain variable domain and a TCR beta-chain variable domain. 【0016】 In some embodiments, the TCR includes alpha-chain variable domains CDR1, CDR2, and CDR3 contained within any one of the alpha-chain variable domain sequences listed in Table 5, and beta-chain variable domains CDR1, CDR2, and CDR3 contained within any one of the beta-chain variable domain sequences listed in Table 5. 【0017】 In some embodiments, the TCR includes an alpha-chain variable domain having an amino acid sequence that has at least 85% amino acid identity with respect to any one entire amino acid sequence among the alpha-chain variable domain amino acid sequences listed in Table 5. 【0018】 In some embodiments, the TCR includes a beta-chain variable domain having an amino acid sequence that has at least 85% amino acid identity with respect to any one entire amino acid sequence among the beta-chain variable domain amino acid sequences listed in Table 5. 【0019】 In some embodiments, the TCR includes (a) an alpha-chain variable domain having an amino acid sequence having at least 85% amino acid identity with respect to any one of the amino acid sequences listed in Table 5, and (b) a beta-chain variable domain having an amino acid sequence having at least 85% amino acid identity with respect to any one of the amino acid sequences listed in Table 5. 【0020】 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: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, and 103; (b) an alpha-chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, and 104; and (c) an alpha-chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, and 105. The invention comprises an in CDR3 domain, a beta-chain variable domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, and 106, a beta-chain variable domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, and 107, and a beta-chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, and 108. 【0021】 In some embodiments, the TCR comprises an amino acid sequence pair of an alpha-chain variable domain / beta-chain variable domain selected from the group consisting of SEQ ID NOs: 217 / 219, 229 / 231, 237 / 239, 241 / 243, and 285 / 287. 【0022】 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: 217 / 219, 221 / 223, 225 / 227, 229 / 231, 233 / 235, 237 / 239, 241 / 243, 245 / 247, 249 / 251, 253 / 255, 257 / 259, 261 / 263, 265 / 267, 269 / 271, 273 / 275, 277 / 279, 281 / 283, and 285 / 287. 【0023】 The present invention also provides a TCR (for example, an isolated TCR or a TCR expressed in an isolated cell) that completes binding to one or more of the TCRs of the present invention. 【0024】 In one embodiment, the present invention provides a T cell receptor (TCR) (e.g., an isolated TCR or a TCR expressed in isolated cells) that specifically binds to an antigen (PRAME) peptide that is preferentially expressed in melanoma and contains the amino acid sequence of SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), which is an HLA-A2 presenting cancer testicular antigen, wherein the TCR comprises an alpha chain variable domain including a complementary determinant region (CDR)3, and CDR3 comprises any one of the alpha chain variable domain CDR3 amino acid sequences shown in Table 6. 【0025】 In one embodiment, the present invention provides a T cell receptor (TCR) (e.g., an isolated TCR or a TCR expressed in isolated cells) that specifically binds to an antigen (PRAME) peptide that is preferentially expressed in melanoma and contains the amino acid sequence of SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), which is an HLA-A2 presenting cancer testicular antigen, wherein the TCR comprises a beta-chain variable domain including a complementary determinant region (CDR)3, and CDR3 comprises any one of the beta-chain variable domain CDR3 amino acid sequences shown in Table 6. 【0026】 In some embodiments, the alpha-chain variable domain further comprises CDR1 and CDR2, where CDR1 comprises one of the alpha-chain variable domain CDR1 amino acid sequences shown in Table 6, and CDR2 independently comprises one of the alpha-chain variable domain CDR2 amino acid sequences shown in Table 6. 【0027】 In some embodiments, the beta-chain variable domain further comprises CDR1 and CDR2, where 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. 【0028】 The TCR may include at least one TCR alpha-chain variable domain and / or at least one TCR beta-chain variable domain, or the TCR may include both a TCR alpha-chain variable domain and a TCR beta-chain variable domain. 【0029】 In some embodiments, the TCR includes alpha-chain variable domains CDR1, CDR2, and CDR3 contained within any one of the alpha-chain variable domain sequences listed in Table 8, and beta-chain variable domains CDR1, CDR2, and CDR3 contained within any one of the beta-chain variable domain sequences listed in Table 8. 【0030】 In some embodiments, the TCR includes an alpha-chain variable domain having an amino acid sequence that has at least 85% amino acid identity with respect to any one entire amino acid sequence among the alpha-chain variable domain amino acid sequences listed in Table 8. 【0031】 In some embodiments, the TCR includes a beta-chain variable domain having an amino acid sequence that has at least 85% amino acid identity with respect to any one entire amino acid sequence among the beta-chain variable domain amino acid sequences listed in Table 8. 【0032】 In some embodiments, the TCR includes (a) an alpha-chain variable domain having an amino acid sequence having at least 85% amino acid identity with respect to any one of the amino acid sequences listed in Table 8, and (b) a beta-chain variable domain having an amino acid sequence having at least 85% amino acid identity with respect to any one of the amino acid sequences listed in Table 8. 【0033】 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: 289, 295, 301, 307, 313, 319, 325, 331, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457, 463, 469, 475, 481, 487, 493, 499, 505, 511, 517, and 523, and (b) SEQ ID NOs: 290, 296, 302, 3 The alpha-chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of 08, 314, 320, 326, 332, 338, 344, 350, 356, 362, 368, 374, 380, 386, 392, 398, 404, 410, 416, 422, 428, 434, 440, 446, 452, 458, 464, 470, 476, 482, 488, 494, 500, 506, 512, 518, and 524, and (c) SEQ ID NOs: 291, 297, 303, 309, 315, 321, 327, 333, 339, 345, 351, 357, 363 (d) Alpha chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of 369, 375, 381, 387, 393, 399, 405, 411, 417, 423, 429, 435, 441, 447, 453, 459, 465, 471, 477, 483, 489, 495, 501, 507, 513, 519, and 525, and (d) SEQ ID NOs: 292, 298, 304, 310, 316, 322, 328, 334, 340, 346, 352, 358, 364, 370, 376, 382, ​​388, 394, 400, 406, 412, 418, 4 A beta-chain variable domain CDR1 having an amino acid sequence selected from the group consisting of 24, 430, 436, 442, 448, 454, 460, 466, 472, 478, 484, 490, 496, 502, 508, 514, 520, and 526, and (e) SEQ ID NOs: 293, 299, 305, 311, 317, 323, 329, 335, 341, 347, 353, 359, 365, 371, 377, 383, 389, 395, 401, 407, 413, 419, 425, 431, 437, 443, 449, 455, 461, 467, 473, 479, 485,(f) A beta-chain variable domain CDR2 having an amino acid sequence selected from the group consisting of 491, 497, 503, 509, 515, 521, and 527, and a beta-chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 294, 300, 306, 312, 318, 324, 330, 336, 342, 348, 354, 360, 366, 372, 378, 384, 390, 396, 402, 408, 414, 420, 426, 432, 438, 444, 450, 456, 462, 468, 474, 480, 486, 492, 498, 504, 510, 516, 522, and 528. 【0034】 In some embodiments, the TCR comprises an amino acid sequence pair of an alpha-chain variable domain / beta-chain variable domain selected from the group consisting of SEQ ID NOs: 825 / 827, 845 / 847, 853 / 855, 857 / 859, 865 / 867, 873 / 875, 885 / 887, 893 / 805, 897 / 899, 901 / 903, 913 / 915, and 925 / 927. 【0035】 In one embodiment, the TCR is sequence numbers 769 / 771, 773 / 775, 777 / 779, 781 / 783, 785 / 787, 789 / 791, 793 / 795, 797 / 799, 801 / 803, 805 / 807, 809 / 811, 813 / 815, 817 / 819, 821 / 823, 825 / 827, 829 / 831, 833 / 835, 837 / 839, 841 / 843, 845 / 847, 849 / 851, 853 / 85 The sequence includes an alpha-chain variable domain / beta-chain variable domain amino acid sequence pair selected from the group consisting of 5, 857 / 859, 861 / 863, 865 / 867, 869 / 871, 873 / 875, 877 / 879, 881 / 883, 885 / 887, 889 / 891, 893 / 805, 897 / 899, 901 / 903, 905 / 907, 909 / 911, 913 / 915, 917 / 919, 921 / 923, and 925 / 927. 【0036】 The present invention also provides a TCR (for example, an isolated TCR or a TCR expressed in an isolated cell) that completes binding to one or more of the TCRs of the present invention. 【0037】 In some embodiments, the TCR of the present invention further includes a detectable portion. 【0038】 The present invention further provides a pharmaceutical composition comprising any of the TCRs of the present invention and a pharmaceutically acceptable carrier or diluent, as well as isolated cells exhibiting any of the TCRs of the present invention. 【0039】 In one embodiment, the present invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding any of the alpha-chain variable domains of the TCRs of the present invention. 【0040】 In another embodiment, the present invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding one of the beta-chain variable domains of the TCRs of the present invention. 【0041】 The present invention also provides a vector comprising the polynucleotide molecule of the present invention, and cells expressing the vector of the present invention. 【0042】 In one embodiment, the present invention provides a method for treating a subject having a PRAME-related disease or disorder. The method comprises administering a therapeutically effective amount of the present invention's TCR (e.g., isolated TCR or TCR expressed in isolated cells), a pharmaceutical composition, or a plurality of cells to a subject, thereby treating the subject. 【0043】 In some embodiments, PRAME-related disease or disorder is PRAME-related cancer. 【0044】 In some embodiments, PRAME-related 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 carcinoma, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, undifferentiated astrocytoma, brain tumor, fallopian tube cancer, primary abdominal 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, colorectal cancer, stomach cancer, gastric cancer These include cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, endometrial cancer, uterine carcinosarcoma, testicular germ cell tumor, uveal melanoma, renal papillary cell carcinoma, renal clear cell carcinoma, thymoma, colon adenocarcinoma, cervical squamous cell carcinoma, cervical tumor, pancreatic adenocarcinoma, liver cancer, hepatocellular carcinoma, mesothelioma, or recurrent non-small cell lung cancer. 【0045】 In some embodiments of the present invention, the TCR of the present invention (e.g., an isolated TCR, or a TCR expressed in isolated cells), a pharmaceutical composition, or a plurality of cells of the present invention are administered to a subject in combination with a second therapeutic agent. 【0046】 TCRs, pharmaceutical compositions, or multiple cells may be administered to a subject subcutaneously, intravenously, intradermally, intraperitoneally, or orally, intramuscularly, or intracranially. 【0047】 In one embodiment, the present invention provides an isolated nucleic acid molecule encoding a T cell receptor (TCR), wherein the TCR specifically binds to an antigen (PRAME) peptide preferentially expressed in melanoma, comprising the amino acid sequence of RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), which is an HLA-A2 presenting cancer testicular antigen, and the TCR comprises an alpha-chain variable domain including a complementary determinant region (CDR)3, wherein CDR3 comprises any one of the alpha-chain variable domain CDR3 amino acid sequences shown in Table 5. 【0048】 In another embodiment, the present invention provides an isolated nucleic acid molecule encoding a T cell receptor (TCR), wherein the TCR specifically binds to an antigen (PRAME) peptide preferentially expressed in melanoma, comprising the amino acid sequence of RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), which is an HLA-A2 presenting cancer testicular antigen, and the TCR comprises a beta-chain variable domain including a complementary determinant region (CDR)3, wherein CDR3 comprises any one of the beta-chain variable domain CDR3 amino acid sequences shown in Table 5. 【0049】 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. 【0050】 In some embodiments, the TCR includes alpha-chain variable domain complementary determination regions (CDRs) 1, 2, and 3 contained within any one of the alpha-chain variable domain sequences listed in Table 5, and beta-chain variable domains CDR1, 2, and 3 contained within any one of the beta-chain variable domain sequences listed in Table 5. 【0051】 In some embodiments, the TCR (e.g., an isolated TCR, or a TCR expressed in an isolated cell) includes an alpha-chain variable domain having an amino acid sequence that has at least 85% amino acid identity with respect to any one of the alpha-chain variable domain amino acid sequences listed in Table 5. 【0052】 In some embodiments, the TCR includes a beta-chain variable domain having an amino acid sequence that has at least 85% amino acid identity with respect to any one entire amino acid sequence among the beta-chain variable domain amino acid sequences listed in Table 5. 【0053】 In some embodiments, the TCR includes (a) an alpha-chain variable domain having an amino acid sequence having at least 85% amino acid identity with respect to any one of the amino acid sequences listed in Table 5, and (b) a beta-chain variable domain having an amino acid sequence having at least 85% amino acid identity with respect to any one of the amino acid sequences listed in Table 5. 【0054】 In some embodiments, 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, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, and 103; (b) an alpha-chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, and 104; and (c) an alpha-chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, and 105. The material comprises a variable a-chain domain CDR3 domain, (d) a variable beta-chain domain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, and 106, (e) a variable beta-chain domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, and 107, and (f) a variable beta-chain domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, and 108. 【0055】 In some embodiments, the TCR comprises an amino acid sequence pair of an alpha-chain variable domain / beta-chain variable domain selected from the group consisting of SEQ ID NOs: 217 / 219, 229 / 231, 237 / 239, 241 / 243, and 285 / 287. 【0056】 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: 217 / 219, 221 / 223, 225 / 227, 229 / 231, 233 / 235, 237 / 239, 241 / 243, 245 / 247, 249 / 251, 253 / 255, 257 / 259, 261 / 263, 265 / 267, 269 / 271, 273 / 275, 277 / 279, 281 / 283, and 285 / 287. 【0057】 In some embodiments, 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: 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, and 211, and (b) SEQ ID NOs: 110, 116, 122, 128, 134, 140, 146, 152, (c) an alpha-chain variable domain CDR2 encoded by a nucleic acid sequence selected from the group consisting of 158, 164, 170, 176, 182, 188, 194, 200, 206, and 212, and (c) an alpha-chain variable domain CDR2 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, and 213 (d) a variable pha-chain domain CDR3, and a variable β-chain domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, and 214, and (e) SEQ ID NOs: 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 1 (f) a beta-chain variable domain CDR2 encoded by a nucleic acid sequence selected from the group consisting of 85, 191, 197, 203, 209, and 215, and (f) a beta-chain variable domain CDR3 encoded by a nucleic acid sequence selected from the group consisting of sequence numbers 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, and 216. 【0058】 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: 218 / 220, 222 / 224, 226 / 228, 230 / 232, 234 / 236, 238 / 240, 242 / 244, 246 / 248, 250 / 252, 254 / 256, 258 / 260, 262 / 264, 266 / 268, 270 / 272, 274 / 276, 278 / 280, 282 / 284, and 286 / 288. 【0059】 The present invention also provides a vector comprising an isolated nucleic acid molecule of the present invention and isolated cells comprising the vector of the present invention. 【0060】 In one embodiment, the present invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding any of the alpha-chain variable domains of the TCRs of the present invention. 【0061】 In another embodiment, the present invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding one of the beta-chain variable domains of the TCRs of the present invention. 【0062】 The present invention also provides a vector comprising the polynucleotide molecule of the present invention, and cells expressing the vector of the present invention. 【0063】 In one embodiment, the present invention provides a method for treating a subject having a PRAME-related disease or disorder. The method comprises administering a therapeutically effective amount of the present invention's TCR (e.g., isolated TCR or TCR expressed in isolated cells), a pharmaceutical composition, or a plurality of cells to a subject, thereby treating the subject. 【0064】 In some embodiments, PRAME-related disease or disorder is PRAME-related cancer. 【0065】 In some embodiments, PRAME-related 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 carcinoma, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, undifferentiated astrocytoma, brain tumor, fallopian tube cancer, primary abdominal 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, colorectal cancer, stomach cancer, gastric cancer These include cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, endometrial cancer, uterine carcinosarcoma, testicular germ cell tumor, uveal melanoma, renal papillary cell carcinoma, renal clear cell carcinoma, thymoma, colon adenocarcinoma, cervical squamous cell carcinoma, cervical tumor, pancreatic adenocarcinoma, liver cancer, hepatocellular carcinoma, mesothelioma, or recurrent non-small cell lung cancer. 【0066】 In some embodiments of the present invention, the TCR of the present invention (e.g., an isolated TCR, or a TCR expressed in isolated cells), a pharmaceutical composition, or a plurality of cells of the present invention are administered to a subject in combination with a second therapeutic agent. 【0067】 TCRs, pharmaceutical compositions, or multiple cells may be administered to a subject subcutaneously, intravenously, intradermally, intraperitoneally, or orally, intramuscularly, or intracranially. 【0068】 In one embodiment, the present invention provides an isolated nucleic acid molecule encoding a T cell receptor (TCR), wherein the TCR specifically binds to a PRAME peptide, an HLA-A2-presenting cancer-testicular antigen, which is preferentially expressed in melanoma and contains the amino acid sequence of SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), and the TCR comprises an alpha-chain variable domain containing a complementary determinant region (CDR)3, wherein CDR3 contains any one of the alpha-chain variable domain CDR3 amino acid sequences shown in Table 8. 【0069】 In another embodiment, the present invention provides an isolated nucleic acid molecule encoding a T cell receptor (TCR), wherein the TCR specifically binds to a PRAME antigen peptide preferentially expressed in melanoma, comprising the amino acid sequence of SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), which is an HLA-A2 presenting cancer testicular antigen, and the TCR comprises a beta-chain variable domain including a complementary determinant region (CDR)3, wherein CDR3 comprises any one of the beta-chain variable domain CDR3 amino acid sequences shown in Table 8. 【0070】 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. 【0071】 In some embodiments, the TCR includes alpha-chain variable domain complementary determination regions (CDRs) 1, 2, and 3 contained within any one of the alpha-chain variable domain sequences listed in Table 8, and beta-chain variable domains CDR1, 2, and 3 contained within any one of the beta-chain variable domain sequences listed in Table 8. 【0072】 In some embodiments, the TCR (e.g., an isolated TCR, or a TCR expressed in an isolated cell) includes an alpha-chain variable domain having an amino acid sequence that has at least 85% amino acid identity with respect to any one of the alpha-chain variable domain amino acid sequences listed in Table 8. 【0073】 In some embodiments, the TCR includes a beta-chain variable domain having an amino acid sequence that has at least 85% amino acid identity with respect to any one entire amino acid sequence among the beta-chain variable domain amino acid sequences listed in Table 8. 【0074】 In some embodiments, the TCR includes (a) an alpha-chain variable domain having an amino acid sequence having at least 85% amino acid identity with respect to any one of the amino acid sequences listed in Table 8, and (b) a beta-chain variable domain having an amino acid sequence having at least 85% amino acid identity with respect to any one of the amino acid sequences listed in Table 8. 【0075】 In some embodiments, 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: 289, 295, 301, 307, 313, 319, 325, 331, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457, 463, 469, 475, 481, 487, 493, 499, 505, 511, 517, and 523, and (b) SEQ ID NOs: 2 The alpha-chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of 90, 296, 302, 308, 314, 320, 326, 332, 338, 344, 350, 356, 362, 368, 374, 380, 386, 392, 398, 404, 410, 416, 422, 428, 434, 440, 446, 452, 458, 464, 470, 476, 482, 488, 494, 500, 506, 512, 518, and 524, and (c) SEQ ID NOs: 291, 297, 303, 309, 315, 321, 327, 333, 339, 345 (d) SEQ ID NOs: 292, 298, 304, 310, 316, 322, 328, 334, 340, 346, 352, 358, 364, 370, 376, 382, ​​388, 394, 405, 411, 417, 423, 429, 435, 441, 447, 453, 459, 465, 471, 477, 483, 489, 495, 501, 507, 513, 519, and 525, and (d) SEQ ID NOs: 292, 298, 304, 310, 316, 322, 328, 334, 340, 346, 352, 358, 364, 370, 376, 382, ​​388, 394, 400, 4 A beta-chain variable domain CDR1 having an amino acid sequence selected from the group consisting of 06, 412, 418, 424, 430, 436, 442, 448, 454, 460, 466, 472, 478, 484, 490, 496, 502, 508, 514, 520, and 526, and (e) SEQ ID NOs: 293, 299, 305, 311, 317, 323, 329, 335, 341, 347, 353, 359, 365, 371, 377, 383, 389, 395, 401, 407, 413, 419, 425, 431, 437, 443, 449, 455, 461, 467,(f) A beta-chain variable domain CDR2 having an amino acid sequence selected from the group consisting of 473, 479, 485, 491, 497, 503, 509, 515, 521, and 527, and a beta-chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 294, 300, 306, 312, 318, 324, 330, 336, 342, 348, 354, 360, 366, 372, 378, 384, 390, 396, 402, 408, 414, 420, 426, 432, 438, 444, 450, 456, 462, 468, 474, 480, 486, 492, 498, 504, 510, 516, 522, and 528. 【0076】 In some embodiments, the TCR comprises an amino acid sequence pair of an alpha-chain variable domain / beta-chain variable domain selected from the group consisting of SEQ ID NOs: 825 / 827, 845 / 847, 853 / 855, 857 / 859, 865 / 867, 873 / 875, 885 / 887, 893 / 805, 897 / 899, 901 / 903, 913 / 915, and 925 / 927. 【0077】 In one embodiment, the TCR is sequence numbers 769 / 771, 773 / 775, 777 / 779, 781 / 783, 785 / 787, 789 / 791, 793 / 795, 797 / 799, 801 / 803, 805 / 807, 809 / 811, 813 / 815, 817 / 819, 821 / 823, 825 / 827, 829 / 831, 833 / 835, 837 / 839, 841 / 843, 845 / 847, 849 / 851, 853 / 85 The sequence includes an alpha-chain variable domain / beta-chain variable domain amino acid sequence pair selected from the group consisting of 5, 857 / 859, 861 / 863, 865 / 867, 869 / 871, 873 / 875, 877 / 879, 881 / 883, 885 / 887, 889 / 891, 893 / 805, 897 / 899, 901 / 903, 905 / 907, 909 / 911, 913 / 915, 917 / 919, 921 / 923, and 925 / 927. 【0078】 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: 529, 535, 541, 547, 553, 559, 565, 571, 577, 583, 589, 595, 601, 607, 613, 619, 625, 631, 637, 643, 649, 655, 661, 667, 673, 679, 685, 691, 697, 703, 709, 715, 721, 727, 733, 739, 745, 751, 757, and 763, and (b) SEQ ID NOs: 530, 5 Alpha-chain variable domain CDR2 encoded by nucleic acid sequences selected from the group consisting of 36, 542, 548, 554, 560, 566, 572, 578, 584, 590, 596, 602, 608, 614, 620, 626, 632, 638, 644, 650, 656, 662, 668, 674, 680, 686, 692, 698, 704, 710, 716, 722, 728, 734, 740, 746, 752, 758, and 764, and (c) Sequence IDs 531, 537, 543, 549, 555, 561, 567, 573, 579, 585, 591 (d) Alpha-chain variable domain CDR3 encoded by nucleic acid sequences selected from the group consisting of 597, 603, 609, 615, 621, 627, 633, 639, 645, 651, 657, 663, 669, 675, 681, 687, 693, 699, 705, 711, 717, 723, 729, 735, 741, 747, 753, 759, and 765, and (d) SEQ ID NOs: 532, 538, 544, 550, 556, 562, 568, 574, 580, 586, 592, 598, 604, 610, 616, 622, 628, 634, 640, 646, 6 (e) a beta-chain variable domain CDR1 encoded by a nucleic acid sequence selected from the group consisting of 52, 658, 664, 670, 676, 682, 688, 694, 700, 706, 712, 718, 724, 730, 736, 742, 748, 754, 760, and 766, and (e) SEQ ID NOs: 533, 539, 545, 551, 557, 563, 569, 575, 581, 587, 593, 599, 605, 611, 617, 623, 629, 635, 641, 647, 653, 659, 665, 671, 677, 683, 689, 695, 701, 707,(f) a beta-chain variable domain CDR2 encoded by a nucleic acid sequence selected from the group consisting of 713, 719, 725, 731, 737, 743, 749, 755, 761, and 767, and a beta-chain variable domain CDR3 encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 534, 540, 546, 552, 558, 564, 570, 576, 582, 588, 594, 600, 606, 612, 618, 624, 630, 636, 642, 648, 654, 660, 666, 672, 678, 684, 690, 696, 702, 708, 714, 720, 726, 732, 738, 744, 750, 756, 762, and 768. 【0079】 In one embodiment, the TCR is sequence numbers 770 / 772, 774 / 776, 778 / 780, 782 / 784, 786 / 788, 790 / 792, 794 / 796, 798 / 800, 802 / 804, 806 / 808, 810 / 812, 814 / 816, 818 / 820, 822 / 824, 826 / 828, 830 / 832, 834 / 836, 838 / 840, 842 / 844, 846 / 848, 850 / 852, 854 / 8 It includes an alpha-chain variable domain / beta-chain variable domain nucleic acid sequence pair selected from the group consisting of 56, 858 / 860, 862 / 864, 866 / 868, 870 / 872, 874 / 876, 878 / 880, 882 / 884, 886 / 888, 890 / 892, 894 / 896, 898 / 900, 902 / 904, 906 / 908, 910 / 912, 914 / 916, 918 / 920, 922 / 924, and 926 / 928. 【0080】 The present invention also provides a vector comprising an isolated nucleic acid molecule of the present invention and isolated cells comprising the vector of the present invention. 【0081】 In one embodiment, the present invention provides a method for treating a subject having a PRAME-related disease or disorder, comprising administering a plurality of cells containing the vector of the present invention to the subject, thereby treating the subject. 【0082】 In some embodiments, PRAME-related disease or disorder is PRAME-related cancer. 【0083】 In some embodiments, PRAME-related 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 carcinoma, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, undifferentiated astrocytoma, brain tumor, fallopian tube cancer, primary abdominal 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, colorectal cancer, stomach cancer, gastric cancer These include cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, endometrial cancer, uterine carcinosarcoma, testicular germ cell tumor, uveal melanoma, renal papillary cell carcinoma, renal clear cell carcinoma, thymoma, colon adenocarcinoma, cervical squamous cell carcinoma, cervical tumor, pancreatic adenocarcinoma, liver cancer, hepatocellular carcinoma, mesothelioma, or recurrent non-small cell lung cancer. 【0084】 In some embodiments, multiple cells are administered to a subject in combination with a second therapeutic agent. 【0085】 The present invention is further described by the following detailed description and drawings. [Modes for carrying out the invention] 【0086】 This invention provides a T cell receptor (TCR) created for the PRAME peptide antigen in association with MHC (HLA-A2). The identified unique TCR sequence exhibits specific binding to the small peptide PRAME present in the groove of the HLA molecule. 【0087】 I. Definition To make the present invention easier to understand, certain terms are defined first. Furthermore, whenever parameter values ​​or ranges of values ​​are enumerated, it should be noted that intermediate values ​​and ranges of the enumerated values ​​are also intended to be part of the present invention. 【0088】 In the following description, for illustrative purposes, certain numbers, materials, and configurations are shown to provide a complete understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be carried out without these specific details. In some cases, well-known properties may be omitted or simplified so as not to obscure the invention. Furthermore, any reference in this specification to phrases such as “one embodiment” or “embodiment” means that certain properties, structures, or features described in relation to an embodiment are included in at least one embodiment of the invention. The appearance of phrases such as “in one embodiment” in various parts of this specification does not necessarily refer to the same embodiment. 【0089】 The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one or more elements. 【0090】 The terms “contains” or “includes” are used herein to refer to compositions, methods, and their respective components that are essential to the disclosure, but may include elements that are not specified as essential. 【0091】 The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any elements not listed in the description of the embodiments. 【0092】 The term "T cell receptor" (TCR), as used herein, refers to an immunoglobulin superfamily member having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail (see, for example, Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p.4:33, 1997), and is capable of specifically binding to antigen peptides bound to MHC receptors. TCRs are found on the surface of cells and generally consist of heterodimers having α and β chains (also known as TCRα and TCRβ, respectively) or γ and δ chains (also known as TCRγ and TCRδ, respectively). Similar to immunoglobulins, the extracellular portion of the TCR chain (e.g., α-chain, β-chain) comprises two immunoglobulin regions adjacent to the cell membrane: a variable region (e.g., TCR variable α-domain or Vα and TCR variable β-domain or Vβ, typically amino acids 1-116 based on Kabat numbering at the N-terminus) and one constant region (e.g., TCR constant domain α or Cα, typically amino acids 117-259 based on Kabat, and TCR constant domain β or Cβ, typically amino acids 117-295 based on Kabat). Also, similar to immunoglobulins, the variable domain contains a complementary determination region (CDR) separated by a framework region (FR). In certain embodiments, the TCR is found on the surface of T cells (or T lymphocytes) and associates with the CD3 complex. The sources of the TCRs of this disclosure may originate from various animal species, such as humans, mice, rats, rabbits, or other mammals. In a preferred embodiment, the source of the TCR of the present invention is a mouse genetically engineered to produce a TCR containing human alpha and beta chains (see, for example, PCT Publication WO2016 / 164492, the entire content of which is incorporated herein by reference). 【0093】 As used herein, the terms “variable region” (variable region of the alpha chain (Vα), variable region of the beta chain (Vβ)) refer to the alpha chain and beta chain, respectively, that are directly involved in the binding of the TCR to the antigen. 【0094】 The "constant regions" of the alpha and beta chains do not directly participate in the binding of the TCR to the antigen, but they exhibit various effector functions. 【0095】 As used herein, the term “antigen” means any substance that causes the immune system to produce antibodies against it or a specific cell-mediated immune response. A disease-associated antigen is any substance associated with any disease that causes the immune system to produce antibodies against it or a specific cell-mediated immune response. 【0096】 The term "PRAME," or "antigen preferentially expressed in melanoma," refers to well-known cancer-testicular antigens (CTAs) that are re-expressed in numerous cancer types. 【0097】 The nucleotide sequences of PRAME are known and can be found, for example, in GenBank accession numbers NM_001291715.2 (sequence number 931), NM_001291716.2 (sequence number 932), NM_001291717.2 (sequence number 933), NM_001291719.2 (sequence number 934), NM_001318126.1 (sequence number 935), NM_001318127.1 (sequence number 936), NM_006115.5 (sequence number 937), NM_206956.3 (sequence number 938), NM_206955.2 (sequence number 939), NM_206954.3 (sequence number 940), and NM_206953.2 (sequence number 941). The amino acid sequence of full-length PRAME is known and can be found, for example, in GenBank under accession numbers NP_001278646.1 (SEQ ID NO: 942), NP_006106.1 (SEQ ID NO: 943), NP_996837.1 (SEQ ID NO: 944), NP_996836.1 (SEQ ID NO: 945), NP_996839.1 (SEQ ID NO: 946), NP_996838.1 (SEQ ID NO: 947), NP_001278644.1 (SEQ ID NO: 948), NP_001305055.1 (SEQ ID NO: 949), NP_001305056.1 (SEQ ID NO: 950), NP_001278648.1 (SEQ ID NO: 951), and NP_001278645.1 (SEQ ID NO: 952). The term "PRAME" includes recombinant PRAME or fragments thereof. This term also encompasses PRAME or fragments thereof conjugated to signal sequences such as histidine tags, mouse or human Fc, or ROR1. In certain embodiments, the term includes PRAME or fragments thereof conjugated to or presented by HLA-A2, in relation to HLA-A2. As used herein, the numbering of specific PRAME amino acid residues within a full-length PRAME sequence corresponds to SEQ ID NO: 944. 【0098】 The term "HLA" refers to the human leukocyte antigen (HLA) system or complex, which is a gene complex that codes for 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) presents peptides from inside the cell. 【0099】 The term "HLA-A" refers to a group of human leukocyte antigens (HLAs) encoded by the HLA-A gene locus. HLA-A is one of the three main types of human MHC class I cell surface receptors. The receptor is a heterodimer, consisting 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. 【0100】 The term "HLA-A2" (which may also be referred to as HLA-A2*01, HLA-A*0201, or HLA-A*02:01) is a specific group of class I major histocompatibility complex (MHC) alleles at one HLA-A locus, where the α chain is encoded by the HLA-A*02 gene and the β chain is encoded by the β2-microglobulin or B2M locus. 【0101】 Terms such as "specifically binds to" or "specifically binds to" mean that the TCR forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1 × 10⁻¹⁶. -6 Less than or equal to M, for example, 1 × 10 -8The TCR can be characterized by an equilibrium dissociation constant of M or less (e.g., a smaller KD indicates a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis and surface plasmon resonance. As described herein, the TCR of the present invention specifically binds to an antigen (PRAME) peptide that is preferentially expressed in melanoma, which is an HLA-A2-presenting cancerous testis, for example, a peptide containing amino acid residues 312-320 or 425-433 of PRAME (e.g., the full-length PRAME sequence of SEQ ID NO: 944). 【0102】 The term “off-target peptide” refers to a peptide that differs from the target peptide (e.g., PRAME 312-320 peptide or PRAME 425-433 peptide) by one, two, three, four, five, or more amino acids. In certain embodiments, the term includes peptides that differ from the target peptide by three or fewer amino acids. For example, in a 9-mer peptide, if one, two, or three amino acids are not identical to those of the target peptide, it is considered an “off-target” peptide. In certain embodiments, amino acid identity is expressed in terms of “degree of similarity” (DoS). If six or more amino acids in a 9-mer peptide are identical, the DoS is 6. In certain embodiments, peptides with a DoS of 6 or less are considered “off-target” peptides. The term “off-target” peptide also refers to a peptide that is similar to the target peptide based on sequence homology, is predicted to bind to HLA-A2, and is contained in proteins expressed in essential normal tissues. Thus, in some embodiments, the TCR of this disclosure is at least 10 times lower than its affinity for binding to the off-target peptide. D It can bind to HLA-A2-presenting PRAME peptides (e.g., peptides containing amino acid residues 312-320 or 425-433 of PRAME) with an affinity corresponding to the value. 【0103】 The term “isolated” refers to a composition, compound, substance, or molecule that has been altered by human intervention from its natural state. For example, a composition or substance present in nature is isolated if it has been altered or removed from its original environment, or both. For instance, a polynucleotide or polypeptide that is naturally present in a living animal is not isolated, but the same polynucleotide or polypeptide that has been separated from its naturally occurring coexisting material is isolated as used herein. More specifically, an isolated TCR can refer to a TCR that has been removed from a cell, for example, a purified TCR. A TCR can also be expressed by an isolated cell, for example, a cell 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). 【0104】 When used herein, the term “recombinant” refers to the TCRs of the present invention that are prepared, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA techniques, including, for example, DNA splicing and transgenic expression. The term also refers to TCRs expressed in non-human mammals (including transgenic non-human mammals, e.g., transgenic mice) or cells (e.g., CHO cells) expression systems, or TCRs isolated from recombinant combinatorial human antibody libraries. 【0105】 As used herein, the terms “polynucleotide” and “nucleic acid molecule” are used interchangeably to refer to nucleotides in polymeric forms of any length. Polynucleotides may include deoxyribonucleotides, ribonucleotides, and / or analogues thereof. Nucleotides may have any three-dimensional structure and may 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 may also include modified nucleic acid molecules (e.g., modified bases, sugars, and / or internucleotide linkers). 【0106】 The term “polypeptide” means any polymer that, regardless of its size, preferably essentially consists of any of the 20 natural amino acids. The term “protein” is often used in relation to relatively large proteins, and “peptide” is often used in relation to small polypeptides, although the use of these terms often overlaps in this art. Unless otherwise specified, the term “polypeptide” generally refers to proteins, polypeptides, and peptides. Generally, useful peptides according to this disclosure, when determined by standard molecular sizing techniques such as centrifugation or SDS-polyacrylamide gel electrophoresis, are generally about 0.1 to 100 kDa or larger, up to about 1000 kDa, preferably about 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30 to 50 kDa. 【0107】 The term "vector" refers to a nucleic acid molecule that can autonomously replicate within a host cell and 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 typically 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. 【0108】 In some embodiments, the TCR of the present invention may be conjugated with a ligand, a detectable portion, or a therapeutic portion ("immunoconjugate"), such as a cytotoxin, an anticancer agent, or any other therapeutic portion useful for treating a disease or condition, including PRAME-related diseases or disorders such as PRAME-related cancer. 【0109】 As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that enables real-time analysis of biomolecular interactions by detecting changes in protein concentration within a biosensor matrix, for example, using BIACORE® systems (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). 【0110】 The term "KD" is K D or K d Also known as KD, it is intended to refer to the equilibrium dissociation constant between a particular biomolecule and its binding partner. KD measurement is particularly useful for evaluating protein-protein interactions, such as in antigen-binding protein-antigen interactions. A smaller KD value indicates a larger (or stronger) binding interaction or affinity between the antigen-binding protein and the antigen (e.g., the target). A larger KD value indicates a weaker binding interaction or affinity between the antigen-binding protein and the antigen. 【0111】 The terms “substantial identity” or “substantial identity,” when referring to a nucleic acid or fragment thereof, indicate that, when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, it has 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 of sequence identity, as considered below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, 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. 【0112】 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 described by Dufresne et al. in 2002 in Nature Biotechnology (vol.20, pp.1269-71) and is used in the GenePAST software (GQ Life Sciences, Inc. Boston, MA). 【0113】 When applied to polypeptides, the term “substantial similarity” or “substantially identical” means that two peptide sequences share at least 90% sequence identity, and more preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity, when optimally aligned by a programmed GAP or BESTFIT using default gap weighting. Preferably, the non-identical residue positions are distinguished by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is replaced by 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 the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the proportion or degree of similarity can be adjusted upward to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331 (which is incorporated herein by reference). Examples of amino acid groups having 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 conserved amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative permutation is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45 (incorporated herein by reference). A “moderately conservative” permutation is any change that has a non-negative value in the PAM250 log-likelihood matrix. 【0114】 Sequence similarity in polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similar measurements assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software can use default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or homologous polypeptides between a wild-type protein and its mutant protein. 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, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment of best overlap regions and percent sequence identity between the query sequence and the search sequence (Pearson (2000) above). Sequences can also be compared using the Smith-Waterman homology search algorithm with affine gap search using a gap start penalty 12 and a gap extension penalty 2 in the BLOSUM62 matrix. Another preferred algorithm for comparing the sequences of the present invention with a database containing numerous sequences derived from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, which uses initial setting parameters. See, for example, 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). 【0115】 "Patient-derived TCRs" are TCRs generated by isolating the alpha and beta chains of PRAME-reactive TCRs isolated from T lymphocytes that mediate tumor regression in vivo in subjects with PRAME-associated cancer. 【0116】 A "ffinity-mature TCR" is a TCR generated by in vitro mutagenesis and selection. For example, mutations can be introduced into the TCR sequence by performing untargeted or targeted (e.g., oligonucleotide-directed) mutagenesis, and the subsequent TCR can be screened for affinity to a target, for example, by using phage display. 【0117】 The phrases "activating a T cell response having a stronger or equal signal-to-noise ratio than that of a patient-derived PRAME-specific TCR" or "activating a T cell response having a stronger or equal signal-to-noise ratio than that of an affinity-matured PRAME-specific TCR" mean, for example, when measured by a luminescence bioassay, an increase in T cell signaling (i.e., about 2x or more), amplification (i.e., about 2x), enhancement (i.e., about 2x), or boost in physiological activity (i.e., about 2x). References to a larger T cell response, or a stronger T cell response or activation signal, may be used interchangeably. Various measurements and assays of T cell response or T cell activation are well known to those skilled in the art. 【0118】 The term "therapeutic dose" refers to the amount administered that produces the desired effect. The exact amount depends on the therapeutic purpose and may be verifiable by those skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). The term "effective dose" is intended to encompass contexts such as a pharmaceutically effective amount or a therapeutically effective amount. For example, in a particular embodiment, the effective dose may achieve a beneficial condition, a beneficial outcome, functional activity in a screening assay, or improvement in a clinical condition. 【0119】 As described herein, the TCRs of the present invention may be “administered” to a subject. “Administering” the TCRs of the present invention includes administering cells expressing the TCRs of the present invention (e.g., effector cells such as T cells), administering nucleic acids expressing the TCRs of the present invention (e.g., vectors expressing such TCRs), and administering polypeptides comprising the TCRs of the present invention, the polypeptides being formatted for such administration (e.g., a bispecific polypeptide comprising a TCR chain and a CD3-binding antibody chain). 【0120】 As used herein, the term “Subject” means an animal, preferably a mammal, that requires improvement, prevention, and / or treatment of a PRAME-related disease or disorder, such as PRAME-related cancer (e.g., PRAME-positive cancer). The term includes human subjects that have, or are at risk of having, a PRAME-related disease or disorder, such as PRAME-related cancer. 【0121】 As used in the present invention, “anticancer agent” means any agent useful for treating, improving, or inhibiting cancer, and includes, but is not limited to, cytotoxins, as well as antimetabolites, alkylating agents, anthracyclines, antibiotics, mitotic inhibitors, procarbazines, hydroxyureas, asparaginases, corticosteroids, cyclophosphamides, mitotanes (O,P'-(DDD)), biologics (e.g., antibodies and interferons), and radiopharmaceuticals. As used in the present invention, “cytotoxin or cytotoxic agent” also refers to chemotherapeutic agents and means any agent that is harmful to cells. Examples include Taxol® (paclitaxel), temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinviastine, coichicin, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogs or homologs. 【0122】 The terms "prevent," "preventing," "prevention," and "preventive treatment" mean reducing the likelihood of developing a disorder or condition in subjects who do not currently have the disorder or condition but are at risk of developing it or are prone to developing it. Prevention does not mean preventing subjects from having had a particular disease or disorder in the past. Prevention may require multiple doses. Prevention may include preventing relapse of a disease in subjects who have been free of all disease symptoms, or preventing relapse in relapsing-remitting diseases. 【0123】 II. PRAME T cells (TCRs) and compositions containing PRAME TCRs T cells, along with other immune cell types (polymorphonuclear, eosinophil, basophil, mast cell, B cell, and NK cell), are a subgroup of cells that constitute the cellular components of the immune system. Under physiological conditions, T cells function in immune surveillance and the elimination of foreign antigens. However, under pathological conditions, there is compelling evidence that T cells play a significant role in the cause and transmission of disease. In these disorders, a breakdown of T cell immune tolerance, whether central or peripheral, is a fundamental process in the cause of autoimmune diseases. 【0124】 T cells bind to epitopes on the surface of antigen-presenting cells with small antigenic determinants associated with the major histocompatibility complex (MHC, mouse) or human leukocyte antigen (HLA, human) complex. T cells bind to these epitopes through the T cell receptor (TCR) complex on the surface of the T cell. The T cell receptor is a heterodimer structure consisting of two types of 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, while 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 have an αβ TCR, while a small number of T cells have a γδ TCR. 【0125】 T cell receptor α and β polypeptides (and similarly, γ and δ polypeptides) are linked to each other via disulfide bonds. Each of the two polypeptides constituting the TCR contains a constant and variable region, a transmembrane domain, and an extracellular domain including 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 complementary determinant regions (CDRs). The TCR is expressed on most T cells in the body and is known to be involved in the recognition of MHC restriction antigens. The TCRα 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 cleavage that can bind to the antigen in association with major histocompatibility complex (MHC) (or HLA in humans). The TCR is a detection molecule with exquisite specificity and, like antibodies, exhibits very high diversity. 【0126】 The general structure of TCR molecules, as well as methods for their preparation and use, including their binding to peptide:major histocompatibility complexes, are disclosed. See, for example, PCT / US98 / 04274, PCT / US98 / 20263, and WO99 / 60120. 【0127】 Non-human animals (e.g., rodents, e.g., mice or rats) can be genetically engineered to express human or humanized T cell receptors (TCRs) containing 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 content of which is incorporated herein by reference). For example, the TCRs of the present invention can be produced using VelociT® mouse technology (Regeneron), which is a genetically modified mouse that enables the production of fully human therapeutic TCRs against tumor and / or viral antigens. Those skilled in the art can obtain mutated TCR sequences through standard mutagenesis techniques in conjunction with the assays described herein and test them for specific binding affinity and / or specificity. Useful mutagenesis techniques known in the art include, but are not limited to, de novo gene synthesis, oligonucleotide-directed mutagenesis, region-directed mutagenesis, linker-scanning mutagenesis, and PCR-directed mutagenesis (see, for example, Sambrook et al. (1989) and Ausubel et al. (1999)). 【0128】 Briefly, in one embodiment, a method for generating a TCR for PRAME 312-320 peptide or PRAME 425-433 peptide may include immunizing a non-human animal (e.g., a rodent, e.g., mouse or rat), such as a genetically engineered non-human animal containing a non-reorganized human TCR variable locus in its genome, with PRAME 312-320 peptide or PRAME 425-433 peptide; initiating an immune response in the animal to the peptide; isolating peptide-responsive T cells from the animal; 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 containing the nucleic acid sequence of the human TCR constant region so that the human TCR variable region is operably linked to the human TCR constant region; and expressing human T cell receptors specific to PRAME 312-320 peptide or PRAME 425-433 peptide, respectively, from the construct. In some embodiments, the steps of isolating 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 containing 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 skilled in the art. 【0129】 In some embodiments, a nucleotide sequence encoding a T cell receptor specific to the antigen of interest is expressed in the cell. In some embodiments, the cells expressing the TCR are selected from CHO, COS, 293, HeLa, PERC.6® cells, etc. 【0130】 In obtaining variant TCR coding sequences, 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 loss or reduction of biological activity. In particular, it is well known that conserved 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 alter protein function. The 20 standard amino acids that make up proteins can be broadly classified 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 within the same amino acid group in a protein with another amino acid is unlikely to have adverse effects on the protein's biological activity. 【0131】 In some embodiments, the TCRs of this disclosure may include CDR sequences having one or more substitutions compared to the CDR sequences in Table 5 or Table 8 (e.g., CDR3 sequences such as VαCDR3 or VβCDR3). For example, the TCRs of this disclosure may include CDR sequences having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions compared to the CDR sequences in Table 5 or Table 8. Generally, the TCRs of the present invention function by binding to HLA-A2 presenting PRAME 312-320 peptides or HLA-A2 presenting PRAME 425-433 peptides. As used herein, an HLA presenting peptide (e.g., an HLA-A2 presenting peptide) may refer to a peptide that binds to human leukocyte antigen (HLA) proteins, e.g., HLA proteins expressed on the surface of cells. Therefore, TCRs that bind to HLA-presenting peptides bind to peptides bound by HLA, and optionally bind to HLA itself. Interaction with HLA can confer specificity to binding to peptides presented by specific HLA. In some embodiments, TCRs bind to isolated HLA-presenting peptides. In some embodiments, TCRs bind to HLA peptides presented on the surface of cells. 【0132】 Generally, the TCR of the present invention can function by binding to HLA-A2-presenting PRAME peptides (e.g., PRAME 312-320 or PRAME 425-433). 【0133】 The present invention includes PRAME TCRs that bind with high specificity to PRAME 312-320 peptides or PRAME 425-433 peptides in association with HLA-A2. In some embodiments, the PRAME TCRs do not bind to the PRAME 312-320 peptides or PRAME 425-433 peptides, or such binding is minimal, in the absence of HLA-A2. Furthermore, in some embodiments, the PRAME TCRs do not bind to off-target peptides, or such binding is minimal, in association with HLA-A2. As used herein, off-target peptides can refer to peptides that differ 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 HLA-A2-presenting PRAME(312-320) peptides or HLA-A2-presenting PRAME(425-433) peptides), b) measuring off-target binding, and c) quantifying the difference between the two (e.g., by calculating a ratio). This ratio can be calculated, for example, by dividing the values ​​obtained in a) and b). Measurement of on-target and off-target binding can be achieved, for example, by measuring the percentage of binding to a peptide / HLA tetramer reagent (e.g., PRAME / HLA tetramer reagent) or by other techniques known in the art.In some embodiments, the on-target binding / off-target binding values ​​of the TCR of this disclosure (e.g., values ​​obtained by dividing the values ​​obtained in a) and b) above) are greater than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 It can be 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 values ​​(e.g., values ​​obtained by dividing the values ​​obtained in a) and b) above) can be approximately 5 to approximately 20, approximately 10 to approximately 30, approximately 20 to approximately 80, approximately 30 to approximately 70, approximately 40 to approximately 60, approximately 50 to approximately 250, approximately 100 to approximately 200, approximately 100 to approximately 1000, approximately 300 to approximately 700, approximately 500 to approximately 1500, approximately 800 to approximately 1200, approximately 900 to approximately 1100, approximately 800 to approximately 1500, approximately 1000 to approximately 1400, or approximately 1100 to approximately 1300. 【0134】 In some embodiments, the present invention provides recombinant antigen-binding proteins (e.g., isolated antigen-binding proteins) that specifically bind to the structural epitopes of HLA-A2-presenting human PRAME(312-320) peptides or HLA-A2-presenting human PRAME(425-433) peptides, wherein the antigen-binding proteins (a) bind to monomeric HLA-A2:PRAME(312-320) peptides or monomeric PRAME(312-320) peptides with a binding-dissociation equilibrium constant (K) less than approximately 20 nM when measured by surface plasmon resonance assay at 25°C.D ) the property of binding with, (b) for the monomeric HLA-A2:PRAME(425 - 433) peptide or the monomeric PRAME(425 - 433) peptide, when measured by surface plasmon resonance assay at 25°C, a binding dissociation equilibrium constant (K D ) the property of binding with, (c) for cells expressing the HLA-A2:PRAME(312 - 320) peptide or cells expressing the PRAME(425 - 433) peptide, when determined by luminescence assay, an EC of less than about 6 nM 50 binds with and does not bind to cells expressing the predicted off-target peptide, (d) for cells expressing the HLA-A2:PRAME(312 - 320) peptide or cells expressing the PRAME(425 - 433) peptide, when determined by luminescence assay, an EC of less than about 1 nM 50 binds with and does not substantially bind to cells expressing the predicted off-target peptide, (e) for cells expressing the HLA-A2:PRAME(312 - 320) peptide or cells expressing the PRAME(425 - 433) peptide, when determined by flow cytometry assay, an EC of less than about 30 nM 50 the property of binding with, (f) for cells expressing the HLA-A2:PRAME(312 - 320) peptide or cells expressing the PRAME(425 - 433) peptide, when determined by flow cytometry assay, an EC of less than about 75 nM 50 the property of binding with, and (g) the property that the conformational epitope contains one or more amino acids of SEQ ID NO: 944, and has a property selected from the group consisting of. 【0135】 In some embodiments, the PRAME TCRs of this disclosure have specific activity or affinity for PRAME(312-320) or PRAME(425-433) as measured by in vitro assays. For example, cells expressing HLA (e.g., T2 cells) can be pulsed with PRAME(312-320) or PRAME(425-433) polypeptides, or off-target polypeptides, thereby inducing the cells to present HLA-binding polypeptides. Alternatively, in addition to using off-target polypeptides as a control, off-target HLAs (HLAs other than those recognized by the TCR of interest) can be used. For example, off-target HLAs can be used to present PRAME peptides and test the specificity of their binding to HLA-A2-presenting PRAME peptides. In addition, the control may be a cell line that does not express either PRAME or the target HLA (e.g., HLA-A2). Cells can be co-cultured with a population of T cells expressing the TCR of interest, and activity is measured as a function of the amount of cytokines (e.g., interferon-γ) produced by the cells. In a particular embodiment, the assay has an effector cell:target cell ratio of 1:1 (1 × 10⁻¹⁰). 5 TCR-expressing T cell population in individual effector cells / 96 wells and 10 -10 The assay may include in vitro co-culture of M peptide-loaded T2 cells and interferon-γ measurement 24 hours after co-culture (e.g., by Meso Scale Discovery (MSD® Sector Imager)). In certain embodiments, the assay may have an effector cell:target cell ratio of 5:1 (2.5 × 10⁻¹⁶). 5 Individual effector cells: 5 x 10 4 This may include in vitro co-culturing of a population of TCR-expressing T cells (individual target cells) with effector cells, and measurement of interferon-γ 24 hours after co-culturing (e.g., by Meso Scale Discovery (MSD® Sector Imager)). 【0136】 The detected increase in cytokines can be used as an indicator of activity. The activity or specificity of the TCR of interest against its target peptide compared to the control (off-target), or the activity or specificity of the TCR of interest against its on-target HLA-binding target peptide compared to the off-target HLA-binding target peptide, can be 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 100 times, 200 times, 300 times or more. It can be 400 times or more, 500 times or more, 600 times or more, 700 times or more, 800 times or more, 900 times or more, 1,000 times or more, 1,500 times or more, 2,000 times or more, 2,500 times or more, 3,000 times or more, 4,000 times or more, 5,000 times or more, 10,000 times or more, 20,000 times or more, 30,000 times or more, 40,000 times or more, 50,000 times or more, 60,000 times or more, 70,000 times or more, 80,000 times or more, 90,000 times or more, or 100,000 times or more. 【0137】 In certain embodiments, the PRAME TCR of this disclosure, when administered prophylactically to a subject in need, may be useful in inhibiting tumor growth or delaying cancer progression, thereby increasing the subject's survival rate. For example, administration of the PRAME TCR of the present invention may result in a reduction of the primary tumor and prevent the development of metastases or secondary tumors. In certain embodiments, the PRAME TCR of the present invention, when administered therapeutically to a subject in need, may be useful in inhibiting tumor growth, thereby increasing the subject's survival rate. For example, administration of a therapeutically effective dose of the PRAME TCR of the present invention to a subject may result in established tumor reduction and disappearance in the subject. 【0138】 In some embodiments, the present invention relates to HLA-A2 presentation PRAME The present invention provides a TCR (e.g., an isolated TCR or a TCR expressed in an isolated cell) that specifically binds to peptides 312-320, and the antigen-binding protein exhibits one or more of the following characteristics: (i) an alpha-chain variable domain containing a complementary determinant region (CDR)3, wherein CDR3 contains one amino acid sequence from any of the alpha-chain variable domain CDR3 amino acid sequences shown in Table 3; (ii) a beta-chain variable domain containing a complementary determinant region (CDR)3, wherein CDR3 contains one amino acid sequence from any of the beta-chain variable domain CDR3 amino acid sequences shown in Table 3; (iii) an alpha-chain variable domain containing CDR1, or a substantially similar sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and independently, an alpha-chain variable domain containing one of the CDR2 amino acid sequences shown in Table 3. (iv) CDR1 of the beta-chain variable domain containing any one of the CDR1 amino acid sequences shown in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and independently, as shown in Table 3 (v) The CDR2 of the beta chain variable domain containing any one of the CDR2 amino acid sequences, or a substantially similar sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, (v) the alpha chain variable domains CDR1, CDR2, and CDR3 contained in any one of the alpha chain variable domain sequences listed in Table 5, or at least 90%, at least 95%, at least 96%, at least 97%, at least 98%,(vi) The entire amino acid sequence of any one of the alpha-chain variable domain amino acid sequences listed in Table 5, and at least the entire amino acid sequence of any one of the alpha-chain variable domain amino acid sequences listed in Table 5, or the substantially similar sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, (vi) the entire amino acid sequence of any one of the alpha-chain variable domain amino acid sequences listed in Table 5, and at least (vii) The alpha chain variable domain has an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or approximately 100% amino acid identity. (viii) (a) an alpha-chain variable domain having an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or approximately 100% amino acid identity with any one amino acid sequence of the alpha-chain variable domain amino acid sequences listed in Table 5, and (b) any one amino acid sequence of the beta-chain variable domain amino acid sequences listed in Table 5 (ix)(a) an alpha-chain variable domain CDR1 domain having an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or approximately 100% amino acid identity with the entire anoic acid sequence, or at least 90%, at least 95%,(b) A substantially similar sequence having at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, (b) an alpha-chain variable domain CDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, and 104, or a substantially similar sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, (c) Alpha chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, and 105, or substantially similar sequences having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, (d) SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, and 1 (e) A beta-chain variable domain CDR1 having an amino acid sequence selected from the group consisting of 06, or a substantially similar sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, (e) a beta-chain variable domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, and 107, or at least 90%, at least 95%, at (f) a substantially similar sequence having 96%, at least 97%, at least 98%, or at least 99% sequence identity, and (f) a beta-chain variable domain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, and 108, or a substantially similar sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity,(x) an alpha-chain variable domain / beta-chain variable domain amino acid sequence pair selected from the group consisting of SEQ ID NOs: 217 / 219, 229 / 231, 237 / 239, 241 / 243, and 285 / 287, or a substantially similar sequence 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: 217 / 219, 221 / 223, 225 / 227, 229 / 231, 233 / 235, 237 / 239, 241 / 243, 245 / 247, 249 / 251, 253 / 255, (xii) an alpha-chain variable domain / beta-chain variable domain amino acid sequence pair selected from the group consisting of 257 / 259, 261 / 263, 265 / 267, 269 / 271, 273 / 275, 277 / 279, 281 / 283, and 285 / 287, or a substantially similar sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, (xii) not specifically binding to cells expressing the predicted off-target peptide as determined by a luminescence assay, but HLA-A2 presenting PRAME, It specifically binds to cells expressing peptides 312–320, and / or (xiii) activates a T cell response approximately twice as large as that of patient-derived PRAME-specific TCRs, as determined by a luminescence bioassay of TCR-mediated T cell signaling (e.g., activating a T cell response approximately twice, three, or four times larger than that of patient-derived PRAME-specific TCRs). 【0139】 In some embodiments, the present invention relates to HLA-A2 presentation PRAME The present invention provides a TCR (e.g., an isolated TCR or a TCR expressed in isolated cells) that specifically binds to peptides 425-433, and the antigen-binding protein exhibits one or more of the following characteristics: (i) an alpha-chain variable domain containing a complementary determinant region (CDR)3, wherein CDR3 contains one amino acid sequence from any of the alpha-chain variable domain CDR3 amino acid sequences shown in Table 6; (ii) a beta-chain variable domain containing a complementary determinant region (CDR)3, wherein CDR3 contains one amino acid sequence from any of the beta-chain variable domain CDR3 amino acid sequences shown in Table 6; (iii) an alpha-chain variable domain containing one of the CDR1 amino acid sequences shown in Table 6, or a substantially similar sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and independently an alpha-chain variable domain containing one of the CDR2 amino acid sequences shown in Table 6. (iv) CDR1 of the beta-chain variable domain containing any one of the CDR1 amino acid sequences shown in Table 6, or a substantially similar sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and independently, as shown in Table 6 (v) The CDR2 of the beta chain variable domain containing any one of the CDR2 amino acid sequences, or a substantially similar sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, (v) the alpha chain variable domains CDR1, CDR2, and CDR3 contained in any one of the alpha chain variable domain sequences listed in Table 8, or at least 90%, at least 95%, at least 96%, at least 97%, at least 98%,(vi) The entire amino acid sequence of any one of the alpha-chain variable domain amino acid sequences listed in Table 8, and at least (vii) The alpha chain variable domain has an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or approximately 100% amino acid identity, and (vii) the entire amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 8 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or approximately 100% amino acid identity. (viii) (a) an alpha-chain variable domain having an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% amino acid identity with any one amino acid sequence of the alpha-chain variable domain amino acid sequences listed in Table 8, and (b) any one amino acid sequence of the beta-chain variable domain amino acid sequences listed in Table 8 (ix)(a)Sequence IDs 289, 295, 301, 307, 313, 319, 325, 331, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457, 463,(b) Alpha-chain variable domain CDR1 domain having an amino acid sequence selected from the group consisting of 469, 475, 481, 487, 493, 499, 505, 511, 517, and 523, or substantially similar sequences having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, (b) Sequence IDs 290, 296, 302, 308, 314, 320, 326, 332, 338, 344, 350, 356, 362, 368, 374, 380, 386, 392, 398, (c) SEQ ID NOs: (c) SEQ ID NOs: 291, 297, 303, 309, 315, 321, 327, 333, Alpha chain variable domain CDR3 domain having an amino acid sequence selected from the group consisting of 339, 345, 351, 357, 363, 369, 375, 381, 387, 393, 399, 405, 411, 417, 423, 429, 435, 441, 447, 453, 459, 465, 471, 477, 483, 489, 495, 501, 507, 513, 519, and 525, or substantially the same 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 similar sequences, (d) SEQ ID NOs: 292, 298, 304, 310, 316, 322, 328, 334, 340, 346, 352, 358, 364, 370, 376, 382, ​​388, 394, 400, 406, 412, 418, 424, 430, 436, 442, 448, 454, 460, 466, 472, 478, 484, 490, 496, 502, 508, 514, 520, and 526, or at least 90%, at least 95%, at least 96%, at least 97%,(e) a substantially similar sequence having at least 98% or at least 99% sequence identity, a beta-chain variable domain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 293, 299, 305, 311, 317, 323, 329, 335, 341, 347, 353, 359, 365, 371, 377, 383, 389, 395, 401, 407, 413, 419, 425, 431, 437, 443, 449, 455, 461, 467, 473, 479, 485, 491, 497, 503, 509, 515, 521, and 527, Or substantially similar sequences having sequence identity of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, as well as (f) Sequence IDs 294, 300, 306, 312, 318, 324, 330, 336, 342, 348, 354, 360, 366, 372, 378, 384, 390, 396, 402, 408, 414, 420, 426, 432, 438, 444, 450, 456, 462, 468, 474, 480, 486, 492, 498, 504, 510, 516, 522, (x) an alpha sequence selected from the group consisting of 528 and 825, or a substantially similar sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, (x) an alpha sequence selected from the group consisting of 825 / 827, 845 / 847, 853 / 855, 857 / 859, 865 / 867, 873 / 875, 885 / 887, 893 / 805, 897 / 899, 901 / 903, 913 / 915, and 925 / 927 (xi) Sequence IDs 769 / 771, 773 / 775, 777 / 779, 781 / 783, 785 / 787, 789 / 791, 793 / 795, 797 / 799, 801 / 803, 805 / 807, 809 / 811, 813 / 815, 817 / 819, 821 / 823, 825 / 827, 829 / 831,From 833 / 835, 837 / 839, 841 / 843, 845 / 847, 849 / 851, 853 / 855, 857 / 859, 861 / 863, 865 / 867, 869 / 871, 873 / 875, 877 / 879, 881 / 883, 885 / 887, 889 / 891, 893 / 805, 897 / 899, 901 / 903, 905 / 907, 909 / 911, 913 / 915, 917 / 919, 921 / 923, and 925 / 927. (xii) containing an alpha-chain variable domain / beta-chain variable domain amino acid sequence pair selected from the group, or a substantially similar sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (xii) not specifically binding to cells expressing the predicted off-target peptide as determined by a luminescence assay, but specifically binding to cells expressing the HLA-A2-presenting PRAME 425-433 peptide; and / or (xiii) activating a T cell response approximately twice as large as that of a patient-derived PRAME-specific TCR as determined by a luminescence bioassay of TCR-mediated T cell signaling (e.g., activating a T cell response approximately twice, three, or four times larger than that of a patient-derived PRAME-specific TCR). 【0140】 The TCR of the present invention may have one or more of the above-described biological features, or any combination thereof. Other biological features of the antigen-binding proteins of the present invention will be apparent to those skilled in the art from the overview of this disclosure, including the practical examples herein. 【0141】 In certain embodiments, the polynucleotide encoding the PRAME TCR described herein is inserted into a vector. As used herein, the term “vector” refers to a vehicle into which a protein-encoding polynucleotide can be covalently inserted such that it results in the expression of that protein and / or the cloning of the polynucleotide. Such a vector may also be referred to as an “expression vector.” An isolated polynucleotide may be inserted into a vector using any preferred method known in the art; for example, but not limited to, the vector may be digested using a suitable restriction enzyme and then ligated with the isolated polynucleotide having matching restriction ends. An expression vector has the ability to incorporate and express heterologous or modified nucleic acid sequences encoding at least a portion of a gene product that can be transcribed in a cell. In most cases, the RNA molecule is then translated into a protein. An expression vector may contain a variety of regulatory sequences, which refer to nucleic acid sequences necessary for the transcription or translation of an operablely ligated coding sequence in a particular host organism. In addition to regulatory sequences that govern transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that perform other functions, which are discussed below. An expression vector may include additional elements; for example, an expression vector may have two replication systems, thus allowing it to be maintained in two organisms, such as human cells for expression and a prokaryotic host for cloning and amplification. 【0142】 Expression vectors may have promoter sequences such as CMV, PGK, and EF1α promoters, ribosome recognition and binding TATA boxes, and necessary 5' upstream and 3' downstream regulatory elements such as 3'UTR AAUAAA transcription termination sequences for efficient gene transcription and translation in their respective host cells. Other suitable promoters include constitutive promoters such as Simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV pre-early promoter, and Rous sarcoma virus promoter. Human gene promoters may also be used, including, but are not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. In certain embodiments, inducible promoters are also intended to be part of a vector expressing a chimeric antigen receptor. This provides a molecular switch that can turn on or off the expression of the polynucleotide sequence of interest. Examples of inductive promoters include, but are not limited to, metallothione promoters, glucocorticoid promoters, progesterone promoters, or tetracycline promoters. 【0143】 Expression vectors may contain additional sequences such as 6× histidine (SEQ ID NO: 954), c-Myc, and a FLAG tag, which are incorporated into the TCR being expressed. Thus, expression vectors may be engineered to include 5' and 3' untranslated regulatory sequences, which can sometimes function as enhancer sequences, promoter regions, and / or terminator sequences that promote or enhance the efficient transcription of the target nucleic acid supported by the expression vector. Expression vectors may also be engineered for replication and / or expression functionality (e.g., transcription and translation) in specific cell types, cell locations, or tissue types. Expression vectors may contain selectable markers for vector maintenance in host or receptor cells. 【0144】 Examples of vectors include plasmids, autonomous replication sequences, and transposition factors. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, bacteriophages such as lambda phages or M13 phages, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), and animal viruses. Examples of animal virus categories useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors include the Lenti-X® Bi-Cisstronic Expression System (Neo) vector (Clontrch), the pClneo vector (Promega) for expression in mammalian cells, and pLenti4 / V5-DEST®, pLenti6 / V5-DEST®, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. The coding sequences of the TCRs disclosed herein can be ligated into such expression vectors for the expression of chimeric proteins in mammalian cells. 【0145】 In certain embodiments, the nucleic acid encoding the TCR of the present invention is provided in the form of a viral vector. The viral vector may be derived from a retrovirus, lentivirus, or foam virus. As used herein, the term “viral vector” refers to a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be packaged into a viral vector particle. The viral vector may contain, instead of non-essential viral genes, the coding sequences of various proteins described herein. The vector and / or particle may be used for the purpose of transferring DNA, RNA, or other nucleic acids into cells, either in vitro or in vivo. Numerous forms of viral vectors are known in the art. 【0146】 In certain embodiments, a viral vector containing the coding sequence of the TCR described herein is a retroviral vector or a lentiviral vector. The term “retroviral vector” refers to a vector containing structural and functional genetic elements primarily derived from retroviruses. The term “lentiviral vector” refers to a vector containing structural and functional genetic elements outside the LTR, primarily derived from lentiviruses. 【0147】 The retroviral vectors for use herein may be derived from any known retrovirus (e.g., type c retroviruses such as Moloney's mouse sarcoma virus (MoMSV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), Supumavirus, Friend, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV)). The “retroviruses” of the present invention also include human T-cell leukemia viruses, HTLV-1 and HTLV-2, as well as lentiviruses of the Retroviridae family, such as human immunodeficiency virus, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV), and other classes of retroviruses. 【0148】 For use herein, lentiviral vectors refer to vectors derived from lentiviruses, a group (or genus) of retroviruses that cause progressively developing diseases. Viruses included in this group include HIV (human immunodeficiency virus, including HIV1 and HIV2), Visna Maedi, canine 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 of Dull et al. and Zufferey et al. (Dull et al., J. Virol., 1998;72:8463-8471 and Zufferey et al.) et al., J. Virol. 1998;72:9873-9880). 【0149】 Retroviral vectors for use in the present invention (i.e., both lentivirals and non-lentivirals) can be generated using standard cloning techniques by combining the desired DNA sequences according to the instructions and adaptations described herein (Current Protocols in Molecular Biology, Ausubel, FM 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 applications WO89 / 07136, WO89 / 02468, WO89 / 05345, and WO92 / 07573). 【0150】 Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) sequences for use in vector generation include genomic RNA and cDNA available from commercial sources, such as the Type Culture Collection (ATCC), Rockville, Md. Sequences can also be chemically synthesized. 【0151】 PRAME TCR expression involves introducing a vector into host cells to enable polypeptide expression within those cells. Expression vectors may contain, but are not limited to, various elements for controlling expression, including, 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, promoter sequences may be selected to promote the transcription of polynucleotides in the vector. Suitable promoter sequences include, but are not limited to, the T7 promoter, T3 promoter, SP6 promoter, β-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. Enhancer sequences may be selected to enhance polynucleotide transcription. Selectable markers may allow selection of host cells into which the vector has been inserted from those without; for example, a selectable marker could be a gene conferring antibiotic resistance. Signal sequences may be selected to allow the expressed polypeptide to be transported outside the host cell. 【0152】 In polynucleotide cloning, a vector is introduced into a host cell (an isolated host cell) to enable the replication of the vector itself, thereby amplifying copies of the polynucleotides it contains. Cloning vectors generally include, but are not limited to, an origin of replication, a promoter sequence, a transcription start sequence, an enhancer sequence, and selectable markers. These elements can be appropriately selected by those skilled in the art. For example, the origin of replication may be selected to promote autonomous replication of the vector in the host cell. 【0153】 In certain embodiments, this disclosure provides isolated host cells containing the vectors provided herein. The vector-containing host cells may be useful in the expression or cloning of polynucleotides contained in the vectors. 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 may include, but are not limited to, Gram-negative or Gram-positive organisms, such as enterobacteria such as Escherichia coli, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Sigella, as well as rods such as B. subtilis and B. licheniformis, Pseudomonas aeruginosa, and Streptomysis. 【0154】 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 may be incorporated into the host cell DNA or maintained outside the chromosome. The nucleic acid may be temporarily maintained or stably introduced. Transfection may be achieved by a variety of means known in the art, including, but not limited to, calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and gene gun. Transduction refers to the delivery of a gene using a viral vector or retroviral vector by means of viral infection rather than transfection. In certain embodiments, a retroviral vector is transduced by packaging the vector into virions before contact with cells. For example, the nucleic acid encoding the PRAME TCR of the present invention, supported by a retroviral vector, can be transduced into cells through infection and proviral incorporation. 【0155】 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 entire genetic material of a cell. The terms “genetically modified cell,” “modified cell,” and “redirected cell” are used interchangeably. 【0156】 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., HLA-A2-presenting PRAME peptide, e.g., amino acid residues 312-320 or 425-433 of PRAME). 【0157】 The present invention provides a method for creating immunoeffector cells expressing TCRs as described herein. In some embodiments, the method involves transfecting or transfecting immunoeffector cells (e.g., immunoeffector cells isolated from a subject such as a subject with PRAME-related disease or disorder) so that the immunoeffector cells express one or more TCRs as described herein. In certain embodiments, immunoeffector cells are isolated from an organism and genetically modified without further in vitro manipulation. Such cells can then be directly re-administered to the organism. In further embodiments, immunoeffector cells are first activated and stimulated to proliferate in vitro and then genetically modified to express TCRs. In this regard, immunoeffector cells can be cultured before or after genetic modification (i.e., transfected or transfected to express TCRs as described herein). 【0158】 Prior to in vitro manipulation or genetic modification of the immunoeffector cells described herein, the cell source can be obtained from a subject. In particular, the immunoeffector cells for use in TCR described herein include T cells. 【0159】 T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic fluid, tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. In certain embodiments, T cells can be obtained from units of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL isolation. In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. Apheresis products typically contain lymphocytes, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets, including T cells. In some embodiments, cells recovered by apheresis can be washed to remove the plasma fraction and to place the cells in a suitable buffer or medium for subsequent processing. In some embodiments of the present invention, cells are washed with PBS. In alternative embodiments, the washing solution may be calcium-deficient and magnesium-deficient, or lack many but not all divalent cations. As will be recognized by those skilled in the art, the washing step can be achieved by methods known to those skilled in the art, such as by using a semi-automatic flow-through centrifuge. After washing, the cells can be resuspended in various biocompatible buffers or other physiological saline solutions, with or without buffer. In certain embodiments, undesirable components of the apheresis sample can be removed in the medium in which the cells are directly resuspended. 【0160】 In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes, for example, by centrifugation using 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 a combination of antibodies against surface markers specific to negatively selected cells. One method for use herein is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry, using a cocktail of monoclonal antibodies against cell surface markers present on 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. 【0161】 PBMCs can be used directly for genetic modification by TCR using the methods described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes can be 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 proliferation. 【0162】 Immune effector cells, such as T cells, can be genetically modified after isolation using known methods, or immune effector cells can be activated and proliferated (or differentiated, in the case of progenitor cells) in vitro before genetic modification. In another embodiment, immune effector cells, such as T cells, are genetically modified with a chimeric antigen receptor as described herein (e.g., transduced with a viral vector containing nucleic acid encoding a TCR), and then activated and proliferated in vitro. Methods for activating and proliferating T cells are known in the art and are described, for example, in U.S. Patents 6,905,874, 6,867,041, 6,797,514, WO2012 / 079000, and U.S.2016 / 0175358. 【0163】 The present invention provides a population of modified immune effector cells for the treatment of PRAME-related diseases or disorders (e.g., cancer), the modified immune effector cells comprising PRAME TCRs as disclosed herein. 【0164】 The TCR-expressing immunoeffector cells prepared as described herein can be used in methods and compositions for adoptive immunotherapy according to known techniques or variations thereof, which will be apparent to those skilled in the art based on this disclosure. See, for example, U.S. Patent Application Publication No. 2003 / 0170238 by Gruenberg et al. See also U.S. Patent No. 4,690,915 by Rosenberg et al. 【0165】 Mai. Pharmaceutical composition The present invention provides therapeutic compositions comprising PRAME TCR of the present invention, or immunoeffector cells comprising PRAME TCR of the present invention. The therapeutic compositions according to the present invention are administered together with a suitable carrier, excipients, and other agents incorporated into the formulation to provide improved transport, delivery, tolerance, etc. Many suitable formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, a collection of formulations known to all pharmaceutical chemists. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicle-containing lipids (cationic or anionic) (such as LIPOFECTIN®), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and carbowax-containing semi-solid mixtures. Powell et al. See also al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311. 【0166】 The frequency and duration of treatment can be adjusted according to the severity of the condition. 【0167】 In certain embodiments, a second or subsequent dose of PRAME TCR or immunoeffector cells containing PRAME TCR may be administered after the initial dose, in an amount approximately the same as or less than the initial dose. 【0168】 In certain circumstances, pharmaceutical compositions can be delivered using a sustained-release system. In some embodiments, a pump may be used. 【0169】 Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal, and intramuscular injection, intravenous infusion, etc. The TCRs, pharmaceutical compositions, and cells described herein may be administered via parenteral administration. The preparations of this disclosure may be prepared by publicly known methods. For example, preparations may be prepared, for example, by dissolving, suspending, or emulsifying the above-mentioned antigen-binding proteins or salts thereof in a sterile aqueous or oily medium conventionally used for injection. Aqueous mediums for injection include, for example, physiological saline, isotonic solutions containing glucose, and other adjuvants, which may be used in combination with suitable 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 mediums include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection prepared in this manner is preferably filled into a suitable ampoule. 【0170】 In some embodiments, TCR-expressing immunoeffector cells are first harvested from their culture medium, and then formulated by washing and concentrating the cells in a therapeutically effective dose in a suitable medium and container system ("pharmaceutically acceptable" carrier). Suitable infusion media may be any isotonic media formulation, typically physiological saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), but 5% dextrose or Ringer's lactate solution in water may also be used. The infusion medium may be supplemented with human serum albumin. 【0171】 The number of therapeutically effective cells in the composition is typically 10 cells. 2 Up to 10 (more than 10) 6 There are 10 cells. 8 1 or 10 9 Contains 10 cells 10It may be more than one cell. The number of cells depends on the intended final use of the composition and the cell types contained therein. 【0172】 The cells may be autologous or heterologous to the patient receiving the therapy. If desired, the therapy may also include the administration of mitogens (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. 【0173】 The TCR-expressing immunoeffector cell population of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2 or other cytokines or cell populations. Briefly, the pharmaceutical composition of the present invention may comprise a TCR-expressing immunoeffector cell population, such as T cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such a composition may comprise a buffer such as neutral buffered saline or phosphate-buffered saline, a carbohydrate such as glucose, mannose, sucrose or dextran, or mannitol, an amino acid such as protein, polypeptide or glycine, an antioxidant, a chelating agent such as EDTA or glutathione, an adjuvant (e.g., aluminum hydroxide), and a preservative. The composition of the present invention is preferably formulated for intravenous administration. 【0174】 IV. Therapeutic use of PRAME TCR or immune effector cells containing PRAME TCR The antitumor immune response induced in a subject by administering a TCR expressing the T cells described herein, using the methods described herein or other methods known in the art, may include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses capable of killing infected cells. Humoral immune responses, primarily mediated by helper T cells capable of activating B cells and thus resulting in antibody production, may also be induced. Various techniques can be used to analyze the types of immune responses induced by the compositions of the present invention, which have been well described in the art, such as 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. 【0175】 Therefore, the PRAME TCR of the present invention is particularly useful for the treatment, prevention, and / or improvement of any disease or disorder associated with or mediated by PRAME. For example, the present invention provides a method for treating a PRAME-related disease or disorder, such as PRAME-associated cancer (e.g., PRAME-positive cancer), by administering the PRAME TCR (or a pharmaceutical composition comprising the PRAME TCR or a plurality of cells comprising the PRAME TCR) described herein to a patient in need of such treatment, and provides the PRAME TCR (or a pharmaceutical composition comprising the PRAME TCR) for use in the treatment of PRAME-associated cancer. The antigen-binding protein of the present invention is useful for the treatment, prevention, and / or improvement of a disease or disorder or condition such as PRAME-associated cancer, and / or improvement of at least one symptom associated with such disease, disorder, or condition. In connection with the therapeutic method described herein, the PRAME TCR (or pharmaceutical composition or plurality of cells) may be administered as monotherapy (i.e., as the sole therapeutic agent) or in combination with one or more additional therapeutic agents (examples thereof are described elsewhere herein). 【0176】 Accordingly, the present invention provides a method for treating an individual diagnosed with, suspected of having, or at risk of developing, a PRAME-related disease or disorder (e.g., PRAME-related cancer), the method comprising administering to the individual a therapeutically effective amount of TCR-expressing immunoeffector cells described herein. 【0177】 In one embodiment, the present invention provides a method for treating a subject diagnosed with PRAME-positive cancer, the method comprising: isolating immune effector cells from the subject diagnosed with PRAME-positive cancer; genetically modifying the immune effector cells using a vector containing nucleic acid encoding the TCR of the present invention to produce a population of modified immune effector cells; and administering the population of modified immune effector cells to the subject. In some embodiments, the immune effector cells include T cells. 【0178】 Methods for administering the cell compositions described herein include any method effective for resulting in the reintroduction of ex vivo genetically modified immune effector cells, which either directly express the TCR of the present invention in a subject or express the TCR upon reintroduction of genetically modified precursor cells of immune effector cells that differentiate into mature immune effector cells upon introduction into a subject. One method involves transducing peripheral blood T cells in vitro with a nucleic acid construct according to the present invention and returning the transduced cells to a subject. 【0179】 In some embodiments of the present invention, the compositions described herein are useful for treating subjects suffering from primary or recurrent cancer, including, but not limited to, PRAME-related cancers. For example, PRAME-related 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 carcinoma, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, undifferentiated astrocytoma, brain tumor, fallopian tube cancer, primary abdominal 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, colorectal cancer, stomach cancer, gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, or recurrent non-small cell lung cancer. In some embodiments, PRAME-associated cancers include 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, endometrial cancer, uterine carcinosarcoma, testicular germ cell tumor, uveal melanoma, renal papillary cell carcinoma, renal clear cell carcinoma, thymoma, colon adenocarcinoma, cervical squamous cell carcinoma, cervical tumor, pancreatic adenocarcinoma, liver cancer, hepatocellular carcinoma, mesothelioma, or myxoid round cell liposarcoma. 【0180】 TCRs can be used to treat the early or late stages of PRAME-associated cancer. In some embodiments, the TCRs of the present invention can be used to treat advanced or metastatic cancer. TCRs are useful in reducing, inhibiting, or shrinking tumor growth. In certain embodiments, treatment with the TCRs of the present invention results in regression of more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the tumor in the subject. In certain embodiments, TCRs can be used to prevent tumor recurrence. In certain embodiments, TCRs are useful in extending progression-free survival or overall survival in subjects with PRAME-associated cancer. In some embodiments, TCRs are useful in reducing toxicity caused by chemotherapy or radiotherapy while maintaining long-term survival in patients with PRAME-associated cancer. 【0181】 One or more TCRs of the present invention may be administered to alleviate, prevent, or reduce the severity of one or more symptoms or conditions of a disease or disorder. 【0182】 It is also intended herein that one or more TCRs of the present invention may be used prophylactically in patients at risk of developing a disease or disorder such as PRAME-related disease or disorder (e.g., PRAME-related cancer). 【0183】 In a further embodiment of the present invention, the TCR is used for the preparation of pharmaceutical compositions for treating patients suffering from PRAME-related diseases or disorders, such as PRAME-related cancers. In another embodiment of the present invention, the TCR is used as an adjunct therapy with any other agent or any other therapy known to those skilled in the art that is useful for treating PRAME-related cancers. 【0184】 Combination therapy may comprise PRAME TCRs of the present invention (e.g., immune effector cells comprising TCRs of the present invention), or pharmaceutical compositions of the present invention, and any additional therapeutic agents that can be advantageously combined with TCRs of the present invention. The TCR of the present invention may be synergistically combined with one or more anticancer agents or therapies used to treat or inhibit PRAME-positive cancers, such as 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 carcinoma, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, undifferentiated astrocytoma, brain tumor, fallopian tube cancer, primary abdominal cancer, advanced solid tumors, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, metastatic solid tumors, colorectal cancer, stomach cancer, gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, or recurrent non-small cell lung cancer. 【0185】 This specification envisions the use of the TCR of the present invention in combination with immunostimulatory and / or supportive immunotherapy to inhibit tumor growth and / or enhance the survival of cancer patients. Immunostimulatory therapy includes direct immunostimulatory therapy that enhances immune cell activity and activates the immune response by either “releasing the brakes” or “stepping on the accelerator” with suppressed immune cells. Examples include targeting other checkpoint receptors, vaccination, and adjuvants. Supportive immunomodalities may increase the antigenicity of tumors by promoting immunogenic cell death, inflammation, or have other indirect effects that promote an anti-tumor immune response. Examples include radiation, chemotherapy, anti-angiogenic agents, and surgery. 【0186】 In various embodiments, one or more TCRs of the present invention are PD-1 inhibitors (e.g., anti-PD-1 antibodies such as nivolumab, pembrolizumab, pidilizumab, BGB-A317, or REGN2810), PD-L1 inhibitors (e.g., anti-PD-L1 antibodies such as avelumab, atezolizumab, durvalumab, MDX-1105, or REGN3504), CTLA-4 inhibitors (e.g., ipilimumab), TIM3 inhibitors, BT LA inhibitors, TIGIT inhibitors, CD47 inhibitors, GITR inhibitors, antagonists of other T cell co-inhibitors or ligands (e.g., antibodies against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), indoleamine-2,3, dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists [e.g., "VEGF-Trap" such as aflibercept or other VEGF inhibitory fusion proteins shown in US7,087,411, or anti-VEGF antibodies or their antigen-binding fragments (e.g., bevacizumab or ranibizumab), or small molecule kinase inhibitors of the VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib)], Ang2 inhibitors (e.g., nesbakumab), transforming growth factor β (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib) Antibodies (e.g., cetuximab), NY-ESO-1 inhibitors (e.g., anti-NY-ESO-1 antibodies), 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., BacillusCalmette-Guerin (cancer vaccine), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), costimulators, bispecific antibodies (e.g., bispecific antibodies that act as costimulators, such as CD3×CD20 bispecific antibodies, PSMA×CD3 bispecific antibodies, or bispecific antibodies that bind to tumor antigens and have costimulatory activity), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxyl It may be used in combination with sorbicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiotherapy, surgery, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines (e.g., IL-2, IL-7, IL-21, and IL-15), antibody-drug conjugates (e.g., anti-CD19-DM4 ADC and anti-DS6-DM4 ADC), anti-inflammatory agents (e.g., corticosteroids, nonsteroidal anti-inflammatory drugs), nutritional supplements (e.g., antioxidants), or any other therapies for treating cancer. In certain embodiments, the TCR of the present invention may be used in combination with cancer vaccines, including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, etc., to enhance the antitumor response. 【0187】 Examples of cancer vaccines that can be used in combination with the TCR of the present invention include the MAGE3 vaccine for melanoma and bladder cancer, the MUC1 vaccine for breast cancer, the EGFRv3 vaccine (e.g., rindopepimt) for brain tumors (including glioblastoma multiforme), ALVAC-CEA (for CEA+ cancer), and the NY-ESO-1 vaccine (e.g., for melanoma). 【0188】 In certain embodiments, PRAME TCR of the present invention may be administered in combination with radiotherapy in a manner that generates a long-lasting antitumor response and / or increases the survival rate of patients with cancer. In some embodiments, PRAME TCR of the present invention may be administered before, simultaneously with, or after radiotherapy to cancer patients. For example, radiotherapy may involve administering one or more doses of PRAME TCR of the present invention to the tumor lesion, followed by one or more subsequent doses. In some embodiments, radiotherapy may be administered locally to the tumor lesion to enhance the local immunogenicity of the patient's tumor (active radiation) and / or kill tumor cells (resectable radiation), followed by systemic administration of PRAME TCR of the present invention. 【0189】 Additional therapeutic agents / components may be administered before, concurrently with, or after the administration of PRAME TCR according to the present invention. For the purposes of this disclosure, such administration schemes are considered to be administration of PRAME TCR "combined" with a second therapeutic agent. 【0190】 Additional therapeutic active ingredients may be administered to a subject before administration of PRAME TCR of the present invention. In other embodiments, additional therapeutic active ingredients may be administered to a subject after administration of PRAME TCR of the present invention. In yet another embodiment, additional therapeutic active ingredients may be administered to a subject simultaneously with administration of PRAME TCR of the present invention. "Simultaneous" administration for the purposes of the present invention includes, for example, administering PRAME TCR and additional therapeutic active ingredients to a subject in a single dosage form (e.g., co-formulation) or administering them to a subject in separate dosage forms (administered within about 30 minutes of each other). If administered in separate dosage forms, each dosage form may be administered via the same route, or each dosage form may be administered via a different route. In any case, administering the components in a single dosage form, in separate dosage forms via the same route, or in separate dosage forms via different routes is all considered "simultaneous" for the purposes of this disclosure. For the purposes of this disclosure, administration of PRAME TCR “before,” “simultaneously with,” or “after” (these terms are defined above) administration of an additional therapeutic active ingredient is considered administration of PRAME TCR “combined” with the additional therapeutic active ingredient. 【0191】 The present invention is further illustrated by the following embodiments, but is not intended to be limiting. All references, patents, and published patent applications cited throughout this application are incorporated herein by reference. [Examples] 【0192】 Example 1. Identification of PRAME-specific T cell receptors VelociT® mice, humanized mice with components of the cellular immune system (see, for example, PCT Publication WO2016 / 164492, the entire contents of which are incorporated herein by reference), were immunized with either the PRAME(312-320) peptide (RLDQLLRHV; SEQ ID NO: 929) or the PRAME(425-433) peptide (SLLQHLIGL; SEQ ID NO: 930), which are specifically presented by human HLA-A2. The immunized mice were diluted in PBS and mixed in equal volumes with an adjuvant (e.g., complete Freund's adjuvant (CFA; Chondrex, Inc.)). Spleen suspensions from mice immunized with wither peptides were obtained separately and dissociated. Erythrocytes were dissolved in ACK lysis buffer (Life Technologies), and splenocytes were suspended in RPMI complete medium. Isolated splenocytes were sorted, and single T cells that bind to either PRAME(312-320) or PRAME(425-433) used for immunization in association with MHC were isolated by fluorescence-activated cell sorting (FACS). The isolated T cells were seeded in 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. Each resulting RT product was then split 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 cells 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 PCR products derived from TCR beta and alpha were cloned into expression vectors containing the beta constant region and the alpha constant region, respectively.Expression vectors expressing full-length beta and alpha chain pairs were transfected into CHO cells, and their binding to commercially available PRAME / HLA tetramer reagents was tested. CHO cells were incubated with soluble HLA-A2:PRAME(312-320) or HLA:A2:PRAME(425-433) (MBL International, Woburn, MA) tetramers and antibodies specific to the mouse TCR constant region (clone H57-597) (Biolegend, San Diego, CA). The samples were then analyzed using LSRFortessaX-20 (BD Biosciences, San Jose, CA). To calculate the percentage of tetramer-positive cells, antigen-positive (Ag+) gating was established using FlowJo (LLC, Ashland, OR) based on negative control TCRs that do not bind to the HLA-A2:PRAME(312-320) or HLA:A2:PRAME(425-433)(MBL International, Woburn, MA) tetramer. All Ag+ TCRs had a FlowJo criterion of ≥1% of cells in Ag+ gating by mean fluorescence intensity (MFI) ≥1000. Ag+ TCRs were determined by next-generation sequencing. The total number of TCRs identified by PRAME(312-320) and expressing the same TCR alpha and beta nucleotide sequences is shown in Table 1 below. The cell frequency, i.e., the percentage of tetramer-positive cells in Ag+ gating (%), represents the TCRs shown in the first column of Table 1. The total number of TCRs identified by PRAME(425-433) and expressing the same TCR alpha and beta nucleotide sequences is shown in Table 2 below. The cell frequency, i.e., the percentage of tetramer-positive cells in Ag+ gating, represents the TCRs shown in the first column of Table 2. 【0193】 Table 3 provides a detailed list of the amino acid sequences of the beta-chain variable domains CDR1, CDR2, and CDR3, as well as the alpha-chain variable domains CDR1, CDR2, and CDR3, of the TCR identified by PRAME(312-320) as described above. Table 4 provides a detailed list of the polynucleotide sequences of the beta-chain variable domains CDR1, CDR2, and CDR3, as well as the alpha-chain variable domains CDR1, CDR2, and CDR3, of the TCR identified by PRAME(312-320) as described above. Table 5 provides the amino acid and nucleotide sequences of the beta-chain and alpha-chain variable regions of the TCR identified by PRAME(312-320). 【0194】 Table 6 provides a detailed list of the amino acid sequences of the beta-chain variable domains CDR1, CDR2, and CDR3, as well as the alpha-chain variable domains CDR1, CDR2, and CDR3, of the TCR identified by PRAME(425-433) as described above. Table 7 provides a detailed list of the polynucleic acid sequences of the beta-chain variable domains CDR1, CDR2, and CDR3, as well as the polynucleic acid sequences of the alpha-chain variable domains CDR1, CDR2, and CDR3, of the TCR identified by PRAME(425-433) as described above. Table 8 provides the amino acid and nucleotide sequences of the beta-chain and alpha-chain variable regions of the TCR identified by PRAME(425-433). 【0195】 Table 9 provides the TCR gene family of isolated TCRs identified by PRAME(312-320) as having alpha-variable and beta-variable regions and binding regions, and Table 11 provides the amino acid sequence identifiers and polynucleotide sequence identifiers of the alpha-variable and beta-variable chains of TCRs and CDRs identified by PRAME(312-320). 【0196】 Table 10 provides the TCR gene family of isolated TCRs identified by PRAME(425-433) as having alpha-variable and beta-variable regions and binding regions, and Table 12 provides the amino acid sequence identifiers and polynucleotide sequence identifiers of the alpha-variable and beta-variable chains of TCRs and CDRs identified by PRAME(425-433). [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 3] [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] Table 5-8 Table 5-9 Table 5-10 Table 5-11 Table 5-12 Table 5-13 Table 6-1 Table 6-2 Table 7-1 Table 7-2 Table 7-3 Table 7-4 Table 7-5 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 8-10 Table 8-11 Table 8-12 Table 8-13 Table 8-14 Table 8-15 Table 8-16 Table 8-17 Table 8-18 Table 8-19 Table 8-20 Table 8-21 Table 8-22 Table 8-23 Table 8-24 Table 8-25 Table 8-26 Table 8-27 Table 9 Table 10-1 Table 10-2 Table 11 Table 12-1 Table 12-2 【0197】 Example 2. Dose-dependent activation of T cell receptors By disrupting the genes, Jurkat cell lines lacking expression of endogenous TCRα and TCRβ were generated. These cells were then manipulated at a genomic landing pad site that allows for single-copy Cre recombinase-mediated insertion of a transgenic TCR construct. Subsequently, an activating protein 1 (AP1) response element-driven luciferase reporter was incorporated into this parental bioassay cell line. Specific TCR bioassay lines were constructed by Cre-mediated insertion of constructs expressing VelociT®-derived TCRα and TCRβ sequences. 【0198】 Jurkat bioassay strains expressing the TCR construct were sorted and homogenized using fluorescence-activated cell sorting (FACS) and then tested with peptide-MHC stimulation assays. HEK293 T cells (HLA-A2*01) were seeded into assay wells containing various dilutions of antigenic PRAME peptides (PRAME425-433; SEQ ID NO: 930) or an unrelated HLA-A2-specific peptide (SLLMWITQC; SEQ ID NO: 953). These dilutions were prepared as shown in Table 13. [Table 13] 【0199】 After a 2-hour incubation, engineered Jurkat cells were added to the wells in a 3:1 Jurkat cell:293T cell ratio and incubated for a further 5 hours. Luciferase reporter activity was determined by measuring the endpoint luminescence output in the assay wells. PRAME-specific TCRs mediated dose-dependent AP1 reporter activation in response to HLA-A2*01 HEK293T cells pulsed with the congener peptide, but this was not observed in cells pulsed with the unrelated peptide. The EC50 data are shown in Table 14 below. [Table 14-1] [Table 14-2] *In the assay, the results did not outperform the negative control or did not respond. 【0200】 Equal portions Those skilled in the art can recognize or confirm, by routine experimentation alone, many equivalents to the specific embodiments of the invention described herein, and such equivalents are intended to be encompassed in the following claims. All references, patents, and published patent applications cited throughout this application are incorporated herein by reference.

Claims

[Claim 1] The invention described in the specification.

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