Preferentially expressed antigen in melanoma (PRAME) t cell receptors and methods of use thereof

Abstract

To provide T cell receptor for the antigen primarily expressed in melanoma (PRAME) and use methods thereof.SOLUTION: The invention provides isolated T cell receptors (TCRs) that specifically bind to an antigen peptide preferentially expressed in melanoma (PRAME) peptide being an HLA presented cancer testis antigen, as well as therapeutic and diagnostic methods using the isolated TCRs. The invention provides T cell receptors (TCRs) that are generated against a PRAME peptide antigen in the context of MHC (HLA-A2). The identified unique TCR sequences show specific binding to a small peptide PRAME presented in the groove of an HLA molecule.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 62 / 965,231, filed on January 24, 2020, the entire content of which is incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing that has been electronically submitted in ASCII format, the entire content of which is incorporated herein by reference. The ASCII copy, created on January 20, 2021, is named 118003-00520_SL.txt and is 461,085 bytes in size.

Background Art

[0003] The T cell receptor (TCR) is a membrane-bound heterodimer that includes α and β chains similar to immunoglobulin variable (V) and constant (C) regions. The TCR α chain includes V-α and C-α chains linked by a covalent bond, while the β chain includes a V-β chain covalently linked to the C-β chain. The V-α and V-β chains form pockets or clefts that can bind to antigens in the context of 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] TCR is a major effector of the immune system with unique advantages as a platform for developing therapeutic agents. Antibody therapeutics are limited to the recognition of pathogens in the blood and extracellular space or protein targets on the cell surface, while 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 cells that recognize and activate the presented antigen, TCR can be involved in controlling various immune responses. For example, T cells are involved in regulating the humoral immune response through inducing the differentiation of B cells into antibody-producing cells. Additionally, activated T cells act to initiate cell-mediated immune responses. Therefore, TCR can recognize additional targets not available to antibodies. In addition, TCR has been reported to mediate cell death, increase the proliferation of B cells, and affect the onset and severity of various disorders including cancer, allergy, viral infections, and autoimmune diseases.

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

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

[0007] The antigen preferentially expressed in melanoma, or PRAME, is a well-known cancer testis antigen (CTA) encoded on the X chromosome. It was first identified as a tumor antigen that can be recognized by HLA-A*24-restricted 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 retinoic acid receptors and can thus confer a growth advantage to cancer cells through this mechanism (see, for example, Epping M.T., et al. (2005) Cell 122:835-847).

[0008] PRAME is abundantly re-expressed by 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. However, in normal healthy adult tissues, PRAME expression is limited to the testis. There is a need in the art that is not met for novel targeting agents based on T cell receptors that specifically bind to the PRAME antigen, as well as methods for making and using such agents in therapeutic and diagnostic settings.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0010] The present invention provides a T cell receptor (TCR) prepared against a PRAME peptide antigen in relation to MHC (HLA-A2). The identified specific TCR sequences show 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 that is preferentially expressed in HLA-A2-presenting melanoma and contains the amino acid sequence of RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), wherein the TCR contains an alpha chain variable domain containing a complementary determining region (CDR) 3, and the CDR3 contains any one of the amino acid sequences of the alpha chain variable domain CDR3 amino acid sequences shown in Table 3. (Item 2) A T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide that is preferentially expressed in HLA-A2-presenting melanoma and contains the amino acid sequence of RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), wherein the TCR contains a beta chain variable domain containing a complementary determining region (CDR) 3, and the CDR3 contains any one of the amino acid sequences 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 contains at least one TCR alpha chain variable domain and / or at least one beta chain variable domain. (Item 4) The TCR according to Item 3, wherein the TCR contains a TCR alpha chain variable domain and a TCR beta chain variable domain. (Item 5) The variable alpha domain further comprises CDR1 and CDR2, wherein CDR1 comprises any one of the alpha chain variable domain CDR1 amino acid sequences shown in Table 3, and CDR2 independently comprises any one of the alpha chain variable domain CDR2 amino acid sequences shown in Table 3. The TCR according to any one of items 1 to 4. (Item 6) The variable beta 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. The TCR according to item 5. (Item 7) Alpha chain variable domain CDR1, CDR2, and CDR3 contained within any one of the alpha chain variable domain sequences listed in Table 5, and beta chain variable domain CDR1, CDR2, and CDR3 contained within any one of the beta chain variable domain sequences listed in Table 5. The TCR according to any one of items 4 to 6. (Item 8) A T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide that is preferentially expressed in HLA-A2-presenting melanoma and contains the amino acid sequence of RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), wherein the TCR contains the 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 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, and the TCR according to any one of items 4 to 11, comprising the same. (Item 13) The TCR according to 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 that is preferentially expressed in HLA-A2-presenting melanoma and contains the amino acid sequence of SLLQHLIGL (SEQ ID NO: 930) (PRAME 425 - 433), wherein the TCR comprises an alpha-chain variable domain containing a complementarity-determining region (CDR) 3, and the CDR3 contains any one of the amino acid sequences of the alpha-chain variable domain CDR3 amino acid sequences shown in Table 6. (Item 15) A T cell receptor (TCR) that specifically binds to a melanoma antigen (PRAME) peptide that is preferentially expressed in HLA-A2-presenting melanoma and contains the amino acid sequence of SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), wherein the TCR contains a variable domain of the beta chain containing complementarity-determining region (CDR) 3, and the CDR3 contains the amino acid sequence of any one of the beta chain variable domain CDR3 amino acid sequences shown in Table 6. (Item 16) The TCR according to item 14 or 15, wherein the variable domain of the alpha chain further contains CDR1 and CDR2, the CDR1 contains any one of the alpha chain variable domain CDR1 amino acid sequences shown in Table 6, and the CDR2 independently contains 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 variable domain of the beta chain further contains CDR1 and CDR2, the CDR1 contains any one of the beta chain variable CDR1 amino acid sequences shown in Table 6, and the CDR2 independently contains any one of the beta chain variable domain CDR2 amino acid sequences shown in Table 6. (Item 18) The TCR according to any one of items 14-17, wherein the TCR contains at least one variable domain of the TCR alpha chain and / or at least one variable domain of the beta chain. (Item 19) The TCR according to item 18, wherein the TCR contains a variable domain of the TCR alpha chain and a variable domain of the TCR beta chain. (Item 20) The TCR according to any one of items 17-19, comprising CDR1, CDR2, and CDR3 of the variable domain of the alpha chain contained within any one of the alpha chain variable domain sequences listed in Table 8, and CDR1, CDR2, and CDR3 of the variable domain of the beta chain 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 a melanoma antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma and contains the amino acid sequence of SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), wherein the TCR contains 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: 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 / 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) The TCR according to any one of items 14 to 21, comprising an alpha-chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha-chain variable domain amino acid sequences listed in Table 8. (Item 23) The TCR according to any one of items 14 to 22, comprising a beta-chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta-chain variable domain amino acid sequences listed in Table 8. (Item 24) (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 8, 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 8, wherein the TCR according to any one of items 14 to 23 is included. (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) An 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 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, and the TCR according to any one of items 17 to 24. (Item 26) A TCR according to item 25, comprising an alpha-chain variable domain / beta-chain variable domain amino acid sequence pair selected from the group consisting of 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 / 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. (Item 27) A TCR that competes in binding to a TCR according to any one of items 1 to 26. (Item 28) A TCR that binds to the same epitope as a TCR according to any one of items 1 to 26. (Item 29) A TCR according to any one of items 1 to 28, further comprising a detectable moiety. (Item 30) An isolated cell presenting a TCR according to any one of items 1 to 29. (Item 31) An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the alpha-chain variable domain of a TCR according to any one of items 1 to 29. (Item 32) An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the beta-chain variable domain of a TCR according to any one of items 1 to 29. (Item 33) A vector comprising the polynucleotide molecule according to item 31 or 32. (Item 34) An isolated cell expressing the vector according to item 33. (Item 35) A pharmaceutical composition comprising the isolated cell according to item 34 and a pharmaceutically acceptable carrier or diluent. (Item 36) A method for treating a subject having a PRAME-related disease or disorder, the method comprising administering to the subject a therapeutically effective amount of the TCR according to any one of items 1 to 29, the pharmaceutical composition according to item 35, or the plurality of isolated cells according to item 30, thereby treating the subject. (Item 37) The method according to item 36, wherein the PRAME-related disease or disorder is a PRAME-related cancer. (Item 38) The PRAME-related cancer is liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin disease, multiple myeloma, metastatic solid tumor, colorectal cancer, stomach cancer, gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, endometrial carcinoma of the uterine corpus, carcinosarcoma of the uterus, testicular germ cell tumor, choroidal melanoma, papillary cell carcinoma of the kidney, clear cell carcinoma of the kidney, thymoma, colon adenocarcinoma, squamous cell carcinoma of the cervix, cervical tumor, pancreatic adenocarcinoma, liver cancer, hepatocellular carcinoma, mesothelioma, or recurrent non-small cell lung cancer, the method according to item 37. (Item 39) The method according to any one of items 36 to 38, wherein the TCR, the pharmaceutical composition, or the plurality of cells is 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 is administered to the subject subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. (Item 41) A polynucleotide molecule encoding a T cell receptor (TCR) that specifically binds to a peptide of an antigen (PRAME) preferentially expressed in HLA-A2-presenting melanoma, said TCR comprising an alpha chain variable domain containing a complementarity-determining region (CDR) 3, and said CDR3 comprising an amino acid sequence of any one of the alpha chain variable domain CDR3 amino acid sequences shown in Table 5. (Item 42) A polynucleotide molecule encoding a T cell receptor (TCR) that specifically binds to a peptide of an antigen (PRAME) preferentially expressed in HLA-A2-presenting melanoma, said TCR comprising a beta chain variable domain containing a complementarity-determining region (CDR) 3, and said CDR3 comprising an amino acid sequence of any one of the beta chain variable domain CDR3 amino acid sequences shown in Table 5. (Item 43) The polynucleotide molecule according to 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 according to item 43, wherein said TCR comprises an alpha chain variable domain complementarity-determining region (CDR) 1, CDR2, and CDR3 contained within any one of the alpha chain variable domain sequences listed in Table 5, and a beta chain variable domain CDR1, CDR2, and CDR3 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 a PRAME peptide preferentially expressed in HLA-A2-presenting melanoma, the peptide containing the amino acid sequence of RLDQLLRHV (SEQ ID NO: 929) (PRAME 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 46) The polynucleotide molecule according to any one of items 41-45, wherein the TCR comprises an alpha chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 5. (Item 47) The polynucleotide molecule according to any one of items 41-46, wherein the TCR comprises a beta chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta chain variable domain amino acid sequences listed in Table 5. (Item 48) The polynucleotide molecule according to any one of items 41-47, wherein the TCR comprises (a) an alpha chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 5, and (b) a beta chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta chain variable domain amino acid sequences listed in Table 5. (Item 49) The TCR has (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 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, and is the polynucleotide molecule according to any one of items 41 to 48. (Item 50) The TCR contains 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, and is the polynucleotide molecule according to item 48. (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) an 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) an 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 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, and is the polynucleotide molecule according to Item 48. (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 comprising the polynucleotide molecule according to any one of items 41 to 52. (Item 54) An isolated cell comprising the vector according to 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 and comprising the amino acid sequence of SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), wherein the TCR comprises an alpha chain variable domain comprising a complementarity-determining region (CDR) 3, and the CDR3 comprises any one of the amino acid sequences of the alpha chain variable domain CDR3 amino acid sequences 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 and comprising the amino acid sequence of SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), wherein the TCR comprises a beta chain variable domain comprising a complementarity-determining region (CDR) 3, and the CDR3 comprises any one of the amino acid sequences of the beta chain variable domain CDR3 amino acid sequences shown in Table 8. (Item 57) The polynucleotide molecule according to 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 a peptide of an antigen (PRAME) preferentially expressed in HLA-A2-presenting melanoma, the peptide containing the amino acid sequence of SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), 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: 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 / 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 59) The polynucleotide molecule according to item 58, wherein the TCR comprises alpha chain variable domain complementarity determining regions (CDR) 1, CDR2, and CDR3 contained within any one of the alpha chain variable domain sequences listed in Table 8 and beta chain variable domain CDR1, CDR2, and CDR3 contained within any one of the beta chain variable domain sequences listed in Table 8. (Item 60) The polynucleotide molecule according to any one of items 55-59, wherein the TCR comprises an alpha chain variable domain having an amino acid sequence having at least 85% amino acid identity to the amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 8. (Item 61) The 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 having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta-chain variable domain amino acid sequences listed in Table 8. (Item 62) The 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 to the entire amino acid sequence of any one of the amino acid sequences of the alpha-chain variable domain amino acid sequences listed in Table 8, and (b) a beta-chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta-chain variable domain amino acid sequences listed in Table 8. (Item 63) 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, (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) an 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 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, and the polynucleotide molecule according to any one of items 55 to 62. (Item 64) The polynucleotide molecule according to item 63, 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: 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 / 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, wherein (a) the 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, (b) the 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) an 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 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, and the polynucleotide molecule according to item 62. (Item 66) The polynucleotide molecule according to item 65, 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: 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, 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. (Item 67) A vector comprising the polynucleotide molecule according to any one of items 55 to 66. (Item 68) An isolated cell comprising the vector according to item 67. (Item 69) A method for treating a subject having a PRAME-related disease or disorder, the method comprising administering to the subject a plurality of cells according to item 54 or 68, thereby treating the subject. (Item 70) The method according to item 69, wherein the PRAME-related disease or disorder is a PRAME-related cancer. (Item 71) The method according to item 70, wherein the PRAME-related cancer is liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin disease, multiple myeloma, metastatic solid tumor, colorectal cancer, stomach cancer, gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, endometrial carcinoma of the uterine corpus, carcinosarcoma of the uterus, testicular germ cell tumor, uveal melanoma, papillary renal cell carcinoma of the kidney, clear cell renal cell carcinoma of the kidney, thymoma, colon adenocarcinoma, squamous cell carcinoma of the uterine cervix, tumor of the uterine cervix, 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 aspect, 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-presented cancer testis antigen, wherein the TCR contains an alpha chain variable domain containing complementarity-determining region (CDR) 3, and CDR3 contains the amino acid sequence of any one of the alpha chain variable domain CDR3 amino acid sequences shown in Table 3.

[0012] In another aspect, the present invention provides a T cell receptor (TCR) (e.g., an isolated TCR or a TCR expressed in an isolated cell) that specifically binds to an antigen (PRAME) peptide that is preferentially expressed in melanoma and that contains the amino acid sequence of RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), which is an HLA-A2-presented testicular antigen. The TCR contains a beta-chain variable domain that includes a complementarity-determining region (CDR) 3, and the CDR3 contains the amino acid sequence of 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 includes CDR1 and CDR2, CDR1 contains any one of the alpha-chain variable domain CDR1 amino acid sequences shown in Table 3, and CDR2 independently contains any 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 includes CDR1 and CDR2, CDR1 contains any one of the beta-chain variable CDR1 amino acid sequences shown in Table 3, and CDR2 independently contains any one of the beta-chain variable domain CDR2 amino acid sequences shown in Table 3.

[0015] The TCR can include at least one TCR alpha-chain variable domain and / or at least one beta-chain variable domain, or the TCR can include a TCR alpha-chain variable domain and a TCR beta-chain variable domain.

[0016] In some embodiments, the TCR includes an alpha-chain variable domain CDR1, CDR2, and CDR3 contained within any one of the alpha-chain variable domain sequences listed in Table 5, and a beta-chain variable domain 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 comprises an alpha chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 5.

[0018] In some embodiments, the TCR comprises a beta chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta chain variable domain amino acid sequences listed in Table 5.

[0019] In some embodiments, the TCR comprises: (a) an alpha chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 5; and (b) a beta chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta chain variable domain 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; (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; 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.

[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 (e.g., an isolated TCR, or a TCR expressed in an isolated cell) that binds to any one or more of the TCRs of the present invention.

[0024] In one aspect, the present invention provides a T cell receptor (TCR) (e.g., an isolated TCR or a TCR expressed in an isolated cell) that specifically binds to an antigen (PRAME) peptide that is preferentially expressed in melanoma and that comprises the amino acid sequence of SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), which is an HLA-A2-presented cancer testis antigen, wherein the TCR comprises an alpha chain variable domain comprising a complementarity-determining region (CDR) 3, and the CDR3 comprises the amino acid sequence of any one of the alpha chain variable domain CDR3 amino acid sequences shown in Table 6.

[0025] In one aspect, the present invention provides a T cell receptor (TCR) (e.g., an isolated TCR or a TCR expressed in an isolated cell) that specifically binds to an antigen (PRAME) peptide that is preferentially expressed in melanoma and that comprises the amino acid sequence of SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), which is an HLA-A2-presented cancer testis antigen, wherein the TCR comprises a beta chain variable domain comprising a complementarity-determining region (CDR) 3, and the CDR3 comprises the amino acid sequence of 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, 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.

[0027] In some embodiments, the beta chain variable domain further comprises CDR1 and CDR2, 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 can comprise at least one TCR alpha chain variable domain and / or at least one beta chain variable domain, or the TCR can comprise a TCR alpha chain variable domain and a TCR beta chain variable domain.

[0029] In some embodiments, the TCR comprises alpha chain variable domain CDR1, CDR2, and CDR3 contained within any one of the alpha chain variable domain sequences listed in Table 8, and beta chain variable domain 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 comprises an alpha chain variable domain having an amino acid sequence with at least 85% amino acid identity to the amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 8.

[0031] In some embodiments, the TCR comprises a beta chain variable domain having an amino acid sequence with at least 85% amino acid identity to the amino acid sequence of any one of the amino acid sequences of the beta chain variable domain amino acid sequences listed in Table 8.

[0032] In some embodiments, the TCR comprises: (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 8; 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 8.

[0033] In some embodiments, the TCR has (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) an 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,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 (f) 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 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: 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 / 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 (e.g., an isolated TCR, or a TCR expressed in an isolated cell) that binds to any one or more of the TCRs of the present invention.

[0037] In some embodiments, the TCR of the present invention further comprises a detectable moiety.

[0038] The present invention further provides a pharmaceutical composition comprising any one of the TCRs of the present invention and a pharmaceutically acceptable carrier or diluent, and an isolated cell presenting any one of the TCRs of the present invention.

[0039] In one aspect, the present invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding an alpha chain variable domain of any one of the TCRs of the present invention.

[0040] In another aspect, the present invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding a beta chain variable domain of any one of the TCRs of the present invention.

[0041] The present invention also provides a vector comprising the polynucleotide molecule of the present invention, and a cell expressing the vector of the present invention.

[0042] In one aspect, the present invention provides a method of treating a subject having a PRAME-related disease or disorder. The method comprises administering to the subject a therapeutically effective amount of the TCR of the present invention (e.g., an isolated TCR, or a TCR expressed in an isolated cell), a pharmaceutical composition, or a plurality of cells, thereby treating the subject.

[0043] In some embodiments, the PRAME-related disease or disorder is a PRAME-related cancer.

[0044] In some embodiments, PRAME-related cancers are liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumors, glioblastoma multiforme, anaplastic astrocytoma, brain tumors, fallopian tube cancer, primary peritoneal cancer, advanced solid tumors, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin disease, multiple myeloma, metastatic solid tumors, colorectal cancer, stomach cancer, gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, endometrial carcinoma of the corpus uteri, carcinosarcoma of the uterus, testicular germ cell tumors, uveal melanoma, papillary renal cell carcinoma of the kidney, clear cell renal cell carcinoma of the kidney, thymoma, colon adenocarcinoma, squamous cell carcinoma of the cervix, cervical tumors, pancreatic adenocarcinoma, liver cancer, hepatocellular carcinoma, mesothelioma, or recurrent non-small cell lung cancer.

[0045] In some embodiments of the invention, the TCR of the invention (e.g., an isolated TCR, or a TCR expressed in isolated cells), pharmaceutical composition, or plurality of cells of the invention are administered to a subject in combination with a second therapeutic agent.

[0046] The TCR, pharmaceutical composition, or plurality of cells can be administered to the subject subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.

[0047] In one aspect, the invention provides an isolated nucleic acid molecule encoding a T cell receptor (TCR), wherein the TCR specifically binds to an antigen (PRAME) peptide that is preferentially expressed in melanoma comprising the amino acid sequence of RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), which is an HLA-A2-presented testicular cancer antigen, and the TCR comprises an alpha chain variable domain comprising a complementarity-determining region (CDR) 3, and the CDR3 comprises the amino acid sequence of any one of the alpha chain variable domain CDR3 amino acid sequences shown in Table 5.

[0048] In another aspect, the present invention provides an isolated nucleic acid molecule encoding a T cell receptor (TCR), wherein the TCR specifically binds to a melanoma antigen (PRAME) peptide preferentially expressed in melanoma, which contains the amino acid sequence of RLDQLLRHV (SEQ ID NO: 929) (PRAME 312-320), which is an HLA-A2-presented cancer testicular antigen, and the TCR contains a variable domain of the beta chain containing a complementarity-determining region (CDR) 3, and the CDR3 contains the amino acid sequence of 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 comprises an alpha chain variable domain complementarity-determining region (CDR) 1, CDR2, and CDR3 contained within any one of the alpha chain variable domain sequences listed in Table 5, and a beta chain variable domain CDR1, CDR2, and CDR3 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) comprises an alpha chain variable domain having an amino acid sequence having at least 85% amino acid identity to the amino acid sequence of any one of the alpha chain variable domain amino acid sequences listed in Table 5.

[0052] In some embodiments, the TCR comprises a beta chain variable domain having an amino acid sequence having at least 85% amino acid identity to the amino acid sequence of any one of the beta chain variable domain amino acid sequences listed in Table 5.

[0053] In some embodiments, the TCR comprises: (a) an alpha chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 5; and (b) a beta chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta chain variable domain 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; (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; 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.

[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; (b) an 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) an 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; and (f) 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.

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

[0060] In one aspect, the invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding an alpha chain variable domain of any of the TCRs of the invention.

[0061] In another aspect, the invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding a beta chain variable domain of any of the TCRs of the invention.

[0062] The invention also provides a vector comprising the polynucleotide molecule of the invention and a cell expressing the vector of the invention.

[0063] In one aspect, the invention provides a method of treating a subject having a PRAME-related disease or disorder. The method comprises administering to the subject a therapeutically effective amount of the TCR of the invention (e.g., an isolated TCR, or a TCR expressed in an isolated cell), a pharmaceutical composition, or a plurality of cells, thereby treating the subject.

[0064] In some embodiments, the PRAME-related disease or disorder is a PRAME-related cancer.

[0065] In some embodiments, PRAME-related cancers are liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumors, glioblastoma multiforme, anaplastic astrocytoma, brain tumors, fallopian tube cancer, primary peritoneal cancer, advanced solid tumors, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin disease, multiple myeloma, metastatic solid tumors, colorectal cancer, stomach cancer, gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, endometrial carcinoma of the uterine corpus, carcinosarcoma of the uterus, testicular germ cell tumors, uveal melanoma, papillary cell carcinoma of the kidney, clear cell carcinoma of the kidney, thymoma, colon adenocarcinoma, squamous cell carcinoma of the cervix, cervical tumors, pancreatic adenocarcinoma, liver cancer, hepatocellular carcinoma, mesothelioma, or recurrent non-small cell lung cancer.

[0066] In some embodiments of the invention, the TCR of the invention (e.g., an isolated TCR, or a TCR expressed in isolated cells), pharmaceutical composition, or plurality of cells of the invention are administered to a subject in combination with a second therapeutic agent.

[0067] The TCR, pharmaceutical composition, or plurality of cells can be administered to the subject subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.

[0068] In one aspect, the invention provides an isolated nucleic acid molecule encoding a T cell receptor (TCR), wherein the TCR specifically binds to an antigen (PRAME) peptide that is preferentially expressed in melanoma comprising the amino acid sequence of SLLQHLIGL (SEQ ID NO: 930) (PRAME 425-433), which is an HLA-A2-presented testicular cancer antigen, and wherein the TCR comprises an alpha chain variable domain comprising a complementarity determining region (CDR) 3, and wherein the CDR3 comprises the amino acid sequence of any one of the alpha chain variable domain CDR3 amino acid sequences shown in Table 8.

[0069] In another aspect, 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 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-presented testicular cancer antigen, and the TCR contains a variable beta domain including a complementarity-determining region (CDR) 3, and the CDR3 contains the amino acid sequence of 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 an alpha chain variable domain complementarity-determining region (CDR) 1, CDR2, and CDR3 contained within any one of the alpha chain variable domain sequences listed in Table 8, and a beta chain variable domain CDR1, CDR2, and CDR3 contained within any one of the beta chain variable domain sequences listed in Table 8.

[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 with at least 85% amino acid identity to the entire amino acid sequence of any one of the alpha chain variable domain amino acid sequences listed in Table 8.

[0073] In some embodiments, the TCR includes 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 beta chain variable domain amino acid sequences listed in Table 8.

[0074] In some embodiments, the TCR comprises: (a) an alpha chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha chain variable domain amino acid sequences listed in Table 8; and (b) a beta chain variable domain having an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta chain variable domain 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; (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) an 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,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 (f) 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 alpha-chain variable domain / beta-chain variable domain amino acid sequence pair 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 comprises an alpha-chain variable domain / beta-chain variable domain amino acid sequence pair selected from the group consisting of 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 / 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.

[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; (b) an 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) an 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,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 (f) 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 comprises an alpha-chain variable domain / beta-chain variable domain nucleic acid sequence pair selected from the group consisting of SEQ ID NOs: 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, 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 the isolated nucleic acid molecule of the present invention and an isolated cell comprising the vector of the present invention.

[0081] In one aspect, the present invention provides a method of treating a subject having a PRAME-related disease or disorder, the method comprising administering to the subject a plurality of cells comprising the vector of the present invention, thereby treating the subject.

[0082] In some embodiments, the PRAME-related disease or disorder is a PRAME-related cancer.

[0083] In some embodiments, the PRAME-related cancer is liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, metastatic solid tumor, colorectal cancer, stomach cancer, gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, endometrial carcinoma of the corpus uteri, carcinosarcoma of the uterus, testicular germ cell tumor, uveal melanoma, papillary renal cell carcinoma of the kidney, clear cell renal cell carcinoma of the kidney, thymoma, colon adenocarcinoma, squamous cell carcinoma of the cervix, cervical tumor, pancreatic adenocarcinoma, liver cancer, hepatocellular carcinoma, mesothelioma, or recurrent non-small cell lung cancer.

[0084] In some embodiments, the plurality of 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.

Mode for Carrying Out the Invention

[0086] The present invention provides a T cell receptor (TCR) generated against a PRAME peptide antigen in relation to MHC (HLA-A2). The identified specific TCR sequences show specific binding to the small peptide PRAME present in the groove of the HLA molecule.

[0087] I. Definitions To make the present invention more readily understandable, certain terms are first defined. Further, it should be noted that whenever a value or range of values of a parameter is recited, intermediate values and ranges within the recited values are also intended to be part of the present invention.

[0088] In the following description, for purposes of explanation, specific numbers, materials, and configurations are set forth in order to provide a thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention may be practiced without these specific details. In some instances, well-known characteristics may be omitted or simplified so as not to obscure the present invention. Further, references to terms such as "one embodiment" or "an embodiment" in this specification mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. The appearances of the phrase "in one embodiment" in various places in this specification are not necessarily all referring to the same embodiment.

[0089] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0090] The terms "comprising" or "including" are used herein to refer to compositions, methods, and their respective components that are essential to the present disclosure, but can include elements not specified as being essential.

[0091] The term "consisting of" refers to the compositions, methods, and their respective components described herein, which exclude any element not recited in the description of the embodiment.

[0092] The term "T cell receptor" (TCR), as used herein, refers to a member of the immunoglobulin superfamily having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail (see, e.g., 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 an antigen peptide bound to an MHC receptor. TCRs are found on the surface of cells and generally consist of a heterodimer having an α chain and a β chain (also known as TCRα and TCRβ, respectively), or a γ chain and a δ chain (also known as TCRγ and TCRδ, respectively). Similar to immunoglobulins, the extracellular portion of a TCR chain (e.g., an α chain, a β chain) is a variable region (e.g., TCR variable α region or Vα and TCR variable β region 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, TCR constant domain β or Cβ, typically amino acids 117-295 based on Kabat), which are two immunoglobulin regions adjacent to the cell membrane. Also similar to immunoglobulins, the variable domain contains complementarity-determining regions (CDRs) separated by framework regions (FRs). In certain embodiments, the TCR is found on the surface of a T cell (or T lymphocyte) and associates with the CD3 complex. The source of the TCRs of the present disclosure can be derived from various animal species such as humans, mice, rats, rabbits, or other mammals. In a preferred embodiment, the source of the TCRs of the present invention is a mouse genetically engineered to produce a TCR comprising a human alpha chain and a beta chain (see, e.g., PCT Publication No. WO2016 / 164492, the entire contents of which are incorporated herein by reference).

[0093] As used herein, the term "variable region" (variable region of the alpha chain (Vα), variable region of the beta chain (Vβ)) means each of the alpha and beta chains that is directly involved in the binding of the TCR to an antigen.

[0094] The "constant regions" of the alpha and beta chains do not directly participate in the binding of the TCR to an antigen, but exhibit various effector functions.

[0095] As used herein, the term "antigen" means any substance that elicits an antibody or specific cell-mediated immune response against it in the immune system. A disease-related antigen is any substance associated with any disease that elicits an antibody or specific cell-mediated response against it in the immune system.

[0096] The term "PRAME", or "antigen preferentially expressed in melanoma", refers to a well-known cancer / testis antigen (CTA) that is re-expressed in a number of cancer types.

[0097] The nucleotide sequences of PRAME are known and can be found, for example, in GenBank accession numbers NM_001291715.2 (SEQ ID NO: 931), NM_001291716.2 (SEQ ID NO: 932), NM_001291717.2 (SEQ ID NO: 933), NM_001291719.2 (SEQ ID NO: 934), NM_001318126.1 (SEQ ID NO: 935), NM_001318127.1 (SEQ ID NO: 936), NM_006115.5 (SEQ ID NO: 937), NM_206956.3 (SEQ ID NO: 938), NM_206955.2 (SEQ ID NO: 939), NM_206954.3 (SEQ ID NO: 940), and NM_206953.2 (SEQ ID NO: 941). The amino acid sequences of full-length PRAME are known and can be found, for example, in GenBank as 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. The term also encompasses, for example, PRAME or fragments thereof conjugated to a signal sequence such as a histidine tag, mouse or human Fc, or ROR1. In certain embodiments, the term includes PRAME or fragments thereof that are linked to or presented by HLA-A2 in the context of an association with HLA-A2. As used herein, the numbering of specific PRAME amino acid residues within the full-length PRAME sequence is relative to SEQ ID NO: 944.

[0098] The term "HLA" refers to the human leukocyte antigen (HLA) system or complex, which is a gene complex encoding major histocompatibility complex (MHC) proteins in humans. These cell surface proteins are involved in the regulation of 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 (HLA) encoded by the HLA-A locus. HLA-A is one of the three major types of human MHC class I cell surface receptors. The receptor is a heterodimer composed of a heavy α-chain and a smaller β-chain. The α-chain is encoded by variant HLA-A genes, and the β-chain (β2-microglobulin) is an invariant β2 microglobulin molecule.

[0100] The term "HLA-A2" (which may also be referred to as HLA-A2*01 or HLA-A*0201 or HLA-A*02:01) is a specific class I major histocompatibility complex (MHC) allele group of the 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 "binds specifically 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 -6 M or less, for example, 1×10 -8It can be characterized by an equilibrium dissociation constant of less than M (e.g., a smaller KD represents a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. 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 testicular cancer, 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 a target peptide (e.g., a PRAME 312-320 peptide or a PRAME 425-433 peptide) by 1, 2, 3, 4, 5, or more amino acids. In certain embodiments, the term includes peptides that differ from the target peptide by 3 or fewer amino acids. For example, in a 9-mer peptide, if 1, 2, or 3 amino acids are not identical to the target peptide, it is considered an "off-target" peptide. In certain embodiments, amino acid identity is expressed in terms of the "degree of similarity" (DoS). If 6 or more amino acids within a 9-mer peptide are identical, the DoS is 6. In certain embodiments, peptides having 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 a protein expressed in essential normal tissues. Thus, in some embodiments, the TCR of the present disclosure binds to an HLA-A2-presenting PRAME peptide (e.g., a peptide containing amino acid residues 312-320 or 425-433 of PRAME) with an affinity corresponding to a K D value that is at least 10-fold lower than its affinity for binding to an off-target peptide.

[0103] The term "isolated" refers to a composition, compound, substance, or molecule that has been changed by human hand from its natural state. For example, a composition or substance that occurs in nature is isolated if it has been changed or removed from its original environment, or both. For instance, a polynucleotide or polypeptide that occurs naturally in a living animal is not isolated, but the same polynucleotide or polypeptide separated from its coexisting materials in its natural state is isolated as the term is used herein. More specifically, an isolated TCR can refer to a TCR that has been removed from a cell, e.g., a purified TCR. A TCR can also be expressed by an isolated cell, e.g., a cell 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] The term "recombinant" as used herein refers to a TCR of the present invention that is made, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA techniques, including, for example, DNA splicing and transfection expression. The term refers to a TCR expressed in a non-human mammalian (including a transgenic non-human mammalian, e.g., a transgenic mouse) or cell (e.g., a CHO cell) expression system, or a TCR isolated from a recombinant combinatorial human antibody library.

[0105] As used herein, the terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to nucleotides in polymeric form of any length. Polynucleotides can include deoxyribonucleotides, ribonucleotides, and / or their analogs. Nucleotides can have any three-dimensional structure and can perform any function, whether known or unknown. The term "polynucleotide" includes, for example, single-stranded, double-stranded, and triple-stranded molecules, genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, antisense molecules, cDNA, recombinant polynucleotides, branched polynucleotides, aptamers, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can also include modified nucleic acid molecules (including, for example, modified bases, sugars, and / or internucleotide linkers).

[0106] The term "polypeptide" is meant to refer to any polymer, preferably consisting essentially of any of the 20 natural amino acids, regardless of its size. The term "protein" is often used in connection with relatively large proteins, and "peptide" is often used in connection with small polypeptides, but the use of these terms often overlaps in the art. The term "polypeptide" generally refers to proteins, polypeptides, and peptides, unless otherwise specified. Generally, useful peptides according to the present disclosure are generally about 0.1 to 100 kDa or more, up to about 1000 kDa, preferably about 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30 - 50 kDa as determined by standard molecular sizing techniques such as centrifugation or SDS-polyacrylamide gel electrophoresis.

[0107] The term "vector" refers to a nucleic acid molecule that can replicate autonomously within a host cell and can accept foreign DNA. A vector carries its own origin of replication, one or more unique recognition sites for restriction endonucleases that can be used for the insertion of commonly selectable markers such as foreign DNA and 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 can be conjugated with a ligand, a detectable moiety, or a therapeutic moiety ("immunoconjugate"), e.g., a cytotoxin, an anti-cancer agent, or any other therapeutic moiety useful for treating a disease or condition including a PRAME-related disease or disorder such as PRAME-related cancer.

[0109] The term "surface plasmon resonance" as used herein refers to an optical phenomenon that enables the analysis of real-time biomolecular interactions by detecting changes in the protein concentration within a biosensor matrix, e.g., using a BIACORE (trademark) system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

[0110] The term "KD" is also known as K D or K d and is intended to refer to the equilibrium dissociation constant of a particular biomolecule and its binding partner. KD measurements are particularly useful for evaluating protein-protein interactions, such as in antigen-binding protein-antigen interactions. The smaller the value of KD, the greater (or stronger) the binding interaction or affinity between the antigen-binding protein and the antigen (e.g., target). The larger the value of KD, the weaker the binding interaction or affinity between the antigen-binding protein and the antigen.

[0111] The terms "substantially identical" or "substantially the same", when referring to a nucleic acid or fragment thereof, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, as considered below, when measured by any well-known algorithm for sequence identity, indicate nucleotide sequence identity of at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain cases, encode a polypeptide having the same or substantially the same amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[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 that described by Dufresne et al. in Nature Biotechnology (vol. 20, pp. 1269-71) in 2002 and used in the software GenePAST (GQ Life Sciences, Inc. Boston, MA).

[0113] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences share at least 90% sequence identity, more preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity when optimally aligned by a program such as GAP or BESTFIT using the default gap weights. Preferably, the non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced 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 change the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331 (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 conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions are any change having a positive value in the PAM250 log-likelihood matrix disclosed by Gonnet et al. (1992) Science 256:1443-45 (incorporated herein by reference). A "moderately conservative" substitution is any change having 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 conservative amino acid substitutions. For example, in GCG software, using the default parameters, it is possible to determine the sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides derived from organisms of different species 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 with the default or recommended parameters, which is a program in GCG version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides the alignment of the best overlapping regions and the percent sequence identity between the query sequence and the search sequence (Pearson (2000) supra). Sequences can also be compared using the Smith-Waterman homology search algorithm with an affine gap search using the BLOSUM62 matrix, a gap start penalty of 12, and a gap extension penalty of 2. Another preferred algorithm when comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using the default 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 TCR" is a TCR generated by isolating the alpha and beta chains of a PRAME-reactive TCR from T lymphocytes isolated in vivo from a subject having a PRAME-related cancer and mediating tumor regression in vivo.

[0116] An "affinity matured TCR" is a TCR generated by in vitro mutagenesis and selection. For example, non-targeted or targeted (e.g., oligonucleotide-directed) mutagenesis can be performed to introduce mutations into the TCR sequence, and then the subsequent TCRs can be screened for affinity to the target, for example, by use of phage display.

[0117] The terms "activating a T cell response having a signal-to-noise ratio greater than or equal to that of a patient-derived PRAME-specific TCR" or "activating a T cell response having a signal-to-noise ratio greater than or equal to that of an affinity matured PRAME-specific TCR" mean, for example, when measured by a luminescence bioassay, i.e., an increase (i.e., about 2-fold or more), amplification (i.e., about 2-fold), enhancement (i.e., about 2-fold), or boost in physiological activity (i.e., about 2-fold) of T cell signaling. Reference to a greater T cell response, or a stronger T cell response or activation signal may be used interchangeably. Various measurements and assays of T cell response or T cell activation are well known to those of skill in the art.

[0118] The phrase "therapeutically effective amount" means an amount administered to produce a desired effect. The exact amount will depend on the purpose of the treatment and will be ascertainable by one of skill in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). The term "effective amount" is intended to encompass contexts such as pharmaceutically effective amount or therapeutically effective amount. For example, in certain embodiments, an effective amount can achieve a beneficial condition, beneficial outcome, functional activity in a screening assay, or improvement in a clinical condition.

[0119] As described herein, the TCRs of the present invention can be "administered" to a subject. "Administering" a TCR of the present invention includes administering cells that express the TCR of the present invention (e.g., effector cells such as T cells), administering nucleic acids that express the TCR of the present invention (e.g., vectors that express such TCRs), and administering polypeptides that include the TCR of the present invention, where the polypeptides are formatted for such administration (e.g., bispecific polypeptides that include TCR chains and CD3-binding antibody chains).

[0120] As used herein, the term "subject" refers to an animal, preferably a mammal, in need of amelioration, prevention, and / or treatment of a PRAME-related disease or disorder such as a PRAME-related cancer (e.g., a PRAME-positive cancer). The term includes human subjects having or at risk of having a PRAME-related disease or disorder such as a PRAME-related cancer.

[0121] As used in the present invention, "anticancer agent" means any agent useful for treating, ameliorating, or inhibiting cancer, including, but not limited to, cytotoxins, as well as antimetabolites, alkylating agents, anthracyclines, antibiotics, mitotic inhibitors, procarbazine, hydroxyurea, asparaginase, corticosteroids, cyclophosphamide, mitotane (O,P'-(DDD)), biologics (e.g., antibodies and interferons), and radiopharmaceuticals. As used in the present invention, "cytotoxin or cytotoxic agent" also refers to a chemotherapeutic agent and means any agent that is harmful to cells. Examples include Taxol® (paclitaxel), temozolomide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, coichicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof.

[0122] Terms such as "prevent", "preventing", "prevention", "preventive treatment" mean reducing the likelihood of developing a disorder or condition in a subject who does not have the disorder or condition but is at risk of developing it or is prone to developing it. Prevention etc. does not mean preventing a subject from having previously suffered from a specific disease or disorder. Prevention may require administration of multiple doses. Prevention can include prevention of recurrence of a disease in a subject from whom all disease symptoms have been eliminated, or prevention of recurrence in a relapsing-remitting disease.

[0123] II. Compositions comprising PRAME T cells (TCR) and PRAME TCR T cells are a subgroup of cells that make up the cellular components of the immune system, along with other immune cell types (polymorphonuclear, eosinophils, basophils, mast cells, B cells, NK cells). Under physiological conditions, T cells function in immune surveillance and elimination of foreign antigens. However, there is compelling evidence that under pathological conditions, T cells play a major role in the cause and spread of diseases. In these disorders, breakdown of either central or peripheral T cell immune tolerance is a fundamental process in the cause of autoimmune diseases.

[0124] T cells bind to epitopes, which are small antigenic determinants on the surface of antigen-presenting cells 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 heterodimeric structure consisting of two chains, an α (alpha) and a β (beta) chain, or a γ (gamma) and a δ (delta) chain. The α chain is encoded by a nucleic acid sequence located within the α locus (on human or mouse chromosome 14), which also encompasses the entire δ locus, and the β chain is encoded by a nucleic acid sequence located within the β locus (on mouse chromosome 6 or human chromosome 7). Most 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 (similarly, γ and δ polypeptides) are linked to each other via disulfide bonds. Each of the two polypeptides that make up the TCR contains an extracellular domain including constant and variable regions, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and cytoplasmic tail are also part of the constant region). The variable region of the TCR determines its antigen specificity and, like immunoglobulins, contains three complementarity-determining regions (CDRs). TCRs are expressed on most T cells in the body and are known to be involved in the recognition of MHC-restricted antigens. The TCR α chain contains Vα and Cα regions linked covalently, while the β chain contains a Vβ region covalently linked to the Cβ region. The Vα and Vβ regions form pockets or clefts that can bind antigens in the context of the major histocompatibility complex (MHC) (or HLA in humans). TCRs are detection molecules with exquisite specificity and, like antibodies, exhibit very high diversity.

[0126] The general structure of TCR molecules, as well as methods of making and using them, have been disclosed, including binding to peptide:major histocompatibility complex. See, for example, PCT / US98 / 04274, PCT / US98 / 20263, WO99 / 60120.

[0127] Non-human animals (e.g., rodents, e.g., mice or rats) can be genetically engineered to express a human or humanized T cell receptor (TCR) comprising a variable domain encoded by at least one human TCR variable region gene segment, as described, for example, in PCT Publication No. WO2016 / 164492, the entire content of which is incorporated herein by reference. For example, the VelociT® mouse technology (Regeneron), a genetically modified mouse that enables the production of fully human therapeutic TCRs against tumor and / or viral antigens, can be used to produce the TCRs of the present invention. One of ordinary skill in the art can, in conjunction with the assays described herein, obtain mutated TCR sequences through standard mutagenesis techniques 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-specific mutagenesis, linker-scanning mutagenesis, and site-directed mutagenesis by PCR (see, for example, Sambrook et al. (1989) and Ausubel et al. (1999)).

[0128] Briefly described, in one embodiment, a method for generating a TCR against a PRAME 312-320 peptide or a PRAME 425-433 peptide includes immunizing a non-human animal (e.g., a rodent, such as a mouse or a rat), such as a genetically engineered non-human animal containing an unrearranged human TCR variable locus in its genome, with a PRAME 312-320 peptide or a PRAME 425-433 peptide; initiating an immune response to the peptide in the animal; isolating T cells reactive to the peptide 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 such that the human TCR variable region is operably linked to the human TCR constant region; and expressing a human T cell receptor specific for a PRAME 312-320 peptide or a PRAME 425-433 peptide, respectively, from the construct. In some embodiments, the steps of isolating the T cells, determining the nucleic acid sequence of the human TCR variable region expressed by the T cells, cloning the human TCR variable region into a nucleotide construct 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 for a target antigen is expressed in a 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, one of ordinary skill in the art will recognize that TCR-derived proteins can be modified by certain amino acid substitutions, additions, deletions, and post-translational modifications without loss or reduction of biological activity. In particular, conservative amino acid substitutions, i.e., substitution of one amino acid with another amino acid of similar size, charge, polarity, and steric structure, are well known to be unlikely to significantly alter protein function. The 20 standard amino acids that are the building blocks of proteins can be broadly grouped 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. The negatively charged (acidic) group includes aspartic acid and glutamic acid. Substitution of one amino acid in a protein with another amino acid within the same group is unlikely to have an adverse effect on the biological activity of the protein.

[0131] In some embodiments, the TCRs of the present disclosure can include CDR sequences (e.g., CDR3 sequences such as VαCDR3 or VβCDR3) having one or more substitutions as compared to the CDR sequences of Table 5 or Table 8. For example, the TCRs of the present disclosure can include CDR sequences having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions as compared to the CDR sequences of Table 5 or Table 8. Generally, the TCRs of the present invention function by binding to HLA-A2-presented PRAME 312-320 peptide or HLA-A2-presented PRAME 425-433 peptide. As used herein, an HLA-presented peptide (e.g., an HLA-A2-presented peptide) can refer to a peptide that binds to a human leukocyte antigen (HLA) protein, e.g., an HLA protein expressed on the surface of a cell. Thus, a TCR that binds to an HLA-presented peptide binds to the peptide bound by the HLA and optionally also binds to the HLA itself. Interaction with the HLA can confer specificity for binding to the peptide presented by a particular HLA. In some embodiments, the TCR binds to an isolated HLA-presented peptide. In some embodiments, the TCR binds to an HLA peptide presented on the surface of a cell.

[0132] Generally, the TCRs of the present invention can function by binding to an HLA-A2-presented PRAME peptide (e.g., PRAME 312-320 or PRAME 425-433).

[0133] The present invention involves a PRAME TCR that binds to PRAME 312-320 peptide or PRAME 425-433 peptide with high specificity in relation to HLA-A2. In some embodiments, the PRAME TCR does not bind to the PRAME 312-320 peptide or the PRAME 425-433 peptide, or such binding is minimal, in the absence of HLA-A2. Further, in some embodiments, the PRAME TCR does not bind to off-target peptides in relation to HLA-A2, or such binding is minimal. As used herein, an off-target peptide can refer to a peptide that differs from the target peptide by 1, 2, 3, 4, 5, or more amino acids. In some embodiments, the binding specificity can be determined by: a) measuring on-target binding (e.g., binding to HLA-A2-presented PRAME(312-320) peptide or HLA-A2-presented PRAME(425-433) peptide); b) measuring off-target binding; and c) quantifying the difference between the two (e.g., by calculating a ratio). This ratio can be calculated, for example, by dividing the values obtained in a) and b). The measurement of on-target binding and off-target binding can be achieved, for example, by measuring the percentage of binding to a peptide / HLA tetramer reagent (e.g., a PRAME / HLA tetramer reagent), or by other techniques known in the art.In some embodiments, the on-target binding / off-target binding value of the TCR of the present disclosure (e.g., the value obtained by excluding the values obtained in a) and b) above) is greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, greater than 10, greater than 11, greater than 12, greater than 13, greater than 14, greater than 15, greater than 16, greater than 17, greater than 18, greater than 19, greater than 20, greater than 21, greater than 22, greater than 23, greater than 24, greater than 25, greater than 26, greater than 27, greater than 28, greater than 29, greater than 30, greater than 35, greater than 40, greater than 45, greater than 50, greater than 55, greater than 60, greater than 65, greater than 70, greater than 75, greater than 80, greater than 85, greater than 90, greater than 95, greater than 100, greater than 110, greater than 120, greater than 130, greater than 140, greater than 150, greater than 160, greater than 170, greater than 180, greater than 190, greater than 200, greater than 225, greater than 250, greater than 275, greater than 300, greater than 325, greater than 350, greater than 375, greater than 400, greater than 425, greater than 450, greater than 475, greater than 500, greater than 550, greater than 600, greater than 650, greater than 700, greater than 750, greater than 800, greater than 850, greater than 900, greater than 950, greater than 1000, greater than 1100, greater than 1200, greater than 1300, greater than 1400, greater than 1500, greater than 1600, greater than 1700, greater than 1800, greater than 1900, or greater than 2000. In some embodiments, the on-target binding / off-target binding value (e.g., the value obtained by excluding the values obtained in a) and b) above) is from about 5 to about 20, from about 10 to about 30, from about 20 to about 80, from about 30 to about 70, from about 40 to about 60, from about 50 to about 250, from about 100 to about 200, from about 100 to about 1000, from about 300 to about 700, from about 500 to about 1500, from about 800 to about 1200, from about 900 to about 1100, from about 800 to about 1500, from about 1000 to about 1400, or from about 1100 to about 1300.

[0134] In some embodiments, the present invention provides a recombinant antigen-binding protein (e.g., an isolated antigen-binding protein) that specifically binds to a conformational epitope of an HLA-A2-presented human PRAME(312-320) peptide or a conformational epitope of an HLA-A2-presented human PRAME(425-433) peptide, wherein the antigen-binding protein has a binding dissociation equilibrium constant (K when measured by surface plasmon resonance assay at 25°C to a monomeric HLA-A2:PRAME(312-320) peptide or a monomeric PRAME(312-320) peptide of less than about 20 nMD ) 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 the property of binding with and not binding 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 the property of binding with and substantially not binding 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 these properties.

[0135] In some embodiments, the PRAME TCR of the present disclosure has specific activity or affinity for PRAME(312-320) or PRAME(425-433) as measured by in vitro assays. For example, cells expressing HLA (such as T2 cells) can be pulsed with PRAME(312-320) or PRAME(425-433) polypeptides, or off-target polypeptides, thereby inducing the cells to present a polypeptide that binds to the HLA. Alternatively, or in addition to using an off-target polypeptide as a control, an off-target HLA (an HLA other than the HLA recognized by the TCR of interest) can be used. For example, an off-target HLA can be used to present PRAME peptides and test the specificity of binding to HLA-A2 presented PRAME peptides. In addition, the control can be a cell line that expresses neither PRAME nor the target HLA (e.g., HLA-A2). The cells can be co-cultured with a T cell population expressing the TCR of interest, and activity is measured as a function of the amount of cytokines (such as interferon-gamma) produced by the cells. In certain embodiments, the assay is performed using effector cells:target cells at a ratio of 1:1 (1×10 5 TCR-expressing T cell populations at 10 effector cells / 96 wells and 10 -10 The assay may include in vitro co-culture of M with peptide-loaded T2 cells and measurement of interferon-γ (e.g., by Meso Scale Discovery (MSD® Sector Imager) 24 hours after co-culture. In a specific embodiment, the assay involves an effector:target cell ratio of 5:1 (2.5×10 5 Effector cells: 5 × 10 4 This may include in vitro co-culture of a TCR-expressing T cell population with effector cells at 100 μg / ml of target cells (100 μg / ml of TCR-expressing T cell population) and measuring interferon-γ (e.g., by Meso Scale Discovery (MSD® Sector Imager)) 24 hours after co-culture.

[0136] The increased amount of cytokine detected can be used as an indicator of activity. The activity or specificity of a TCR of interest against its target peptide can be 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 15-fold or more, 20-fold or more, 30-fold or more, 40-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 400-fold or more, 500-fold or more, 600-fold or more, 700-fold or more, 800-fold or more, 900-fold or more, 1,000-fold or more, 1,500-fold or more, 2,000-fold or more, 2,500-fold or more, 3,000-fold or more, 4,000-fold or more, 5,000-fold or more, 10,000-fold or more, 20,000-fold or more, 30,000-fold or more, 40,000-fold or more, 50,000-fold or more, 60,000-fold or more, 70,000-fold or more, 80,000-fold or more, 90,000-fold or more, or 100,000-fold or more compared to a control (off-target), or the activity or specificity of a TCR of interest against its on-target HLA-binding target peptide compared to an off-target HLA-binding target peptide.

[0137] In certain embodiments, the PRAME TCRs of the disclosure are useful for inhibiting tumor growth or delaying cancer progression and increasing the survival rate of a subject when prophylactically administered to a subject in need thereof. For example, administration of the PRAME TCRs of the invention can result in shrinkage of a primary tumor and prevent the occurrence of metastases or secondary tumors. In certain embodiments, the PRAME TCRs of the invention are useful for inhibiting tumor growth and increasing the survival rate of a subject when therapeutically administered to a subject in need thereof. For example, administration of a therapeutically effective amount of the PRAME TCRs of the invention to a subject can result in shrinkage and disappearance of an established tumor in the subject.

[0138] In some embodiments, the present invention provides a TCR that specifically binds to an HLA-A2-presented PRAME 312-320 peptide (e.g., an isolated TCR or a TCR expressed in an isolated cell), wherein the antigen-binding protein exhibits one or more of the following characteristics: (i) an alpha-chain variable domain comprising a complementarity-determining region (CDR) 3, wherein the CDR3 comprises an amino acid sequence of any one of the alpha-chain variable domain CDR3 amino acid sequences shown in Table 3; (ii) a beta-chain variable domain comprising a complementarity-determining region (CDR) 3, wherein the CDR3 comprises an amino acid sequence of any one of the beta-chain variable domain CDR3 amino acid sequences shown in Table 3; (iii) CDR1 of an alpha-chain variable domain comprising 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 thereto, and, independently, CDR2 of an alpha-chain variable domain comprising any one of the CDR2 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 thereto; (iv) CDR1 of a beta-chain variable domain comprising 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 thereto, and, independently, CDR2 of a beta-chain variable domain comprising any one of the CDR2 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 thereto; (v) CDR1, CDR2, and CDR3 of an alpha-chain variable domain 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%,or a substantially similar sequence having at least 99% sequence identity, and the CDR1, CDR2, and CDR3 of the beta-chain variable domain contained in any one of the beta-chain variable domain sequences listed in Table 5, 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; (vi) having an alpha-chain variable domain having an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha-chain variable domain amino acid sequences listed in Table 5; (vii) having a beta-chain variable domain having an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the beta-chain variable domain amino acid sequences listed in Table 5; (viii) (a) having an alpha-chain variable domain having an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the alpha-chain variable domain amino acid sequences listed in Table 5, and (b) having a beta-chain variable domain having an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences of the 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 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, or at least 90%, at least 95%,its substantially similar sequences 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 its 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, (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, or its 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) 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, or its 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, (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 its 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, 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 its 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,(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 thereto; (xi) 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, 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 thereto; (xii) not specifically binding to cells expressing a predicted off-target peptide as determined by a luminescence assay, but specifically binding to cells expressing the HLA-A2-presented PRAME 312-320 peptide; and / or (xiii) activating a T cell response that is approximately 2-fold greater (e.g., activating a T cell response that is approximately 2-fold greater, or approximately 3-fold greater, or approximately 4-fold greater) than a patient-derived PRAME-specific TCR as determined by a luminescence bioassay of TCR-mediated T cell signaling.,

[0139] In some embodiments, the present invention provides a TCR that specifically binds to an HLA-A2-presented PRAME 425-433 peptide (e.g., an isolated TCR or a TCR expressed in an isolated cell), wherein the antigen-binding protein exhibits one or more of the following characteristics: (i) an alpha-chain variable domain comprising a complementarity-determining region (CDR) 3, wherein the CDR3 comprises an amino acid sequence of any one of the alpha-chain variable domain CDR3 amino acid sequences shown in Table 6; (ii) a beta-chain variable domain comprising a complementarity-determining region (CDR) 3, wherein the CDR3 comprises an amino acid sequence of any one of the beta-chain variable domain CDR3 amino acid sequences shown in Table 6; (iii) CDR1 of an alpha-chain variable domain comprising 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 thereto, and, independently, CDR2 of an alpha-chain variable domain comprising any one of the CDR2 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 thereto; (iv) CDR1 of a beta-chain variable domain comprising 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 thereto, and, independently, CDR2 of a beta-chain variable domain comprising any one of the CDR2 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 thereto; (v) CDR1, CDR2, and CDR3 of an alpha-chain variable domain 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%,or a substantially similar sequence having at least 99% sequence identity, and the CDR1, CDR2, and CDR3 of the beta-chain variable domain contained in any one of the beta-chain variable domain sequences listed in Table 8, 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; (vi) 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 the entire amino acid sequence of any one of the amino acid sequences of the alpha-chain variable domain amino acid sequences listed in Table 8; (vii) a beta-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 the entire amino acid sequence of any one of the amino acid sequences of the beta-chain variable domain amino acid sequences listed in Table 8; (viii) (a) 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 the entire amino acid sequence of any one of the amino acid sequences of the alpha-chain variable domain amino acid sequences listed in Table 8, and (b) a beta-chain variable domain having an amino acid sequence 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 the entire amino acid sequence of any one of the amino acid sequences of the beta-chain variable domain amino acid sequences listed in Table 8; (ix) (a) SEQ ID NO: 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,An 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 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 thereto, (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, 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 thereto, (c) an 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, 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 thereto, (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, or at least 90%, at least 95%, at least 96%, at least 97%,its substantially similar sequences having 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: 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 its 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, and (f) 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, or its 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, (x) an alpha-chain variable domain / beta-chain variable domain amino acid sequence pair 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, or its 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, (xi) 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,An alpha-chain variable domain / beta-chain variable domain amino acid sequence pair selected from the group consisting of 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, 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 thereto, (xii) when determined by a luminescence assay, specifically binds to cells expressing HLA-A2-presented PRAME 425-433 peptide but does not specifically bind to cells expressing a predicted off-target peptide, and / or (xiii) when determined by a luminescence bioassay of TCR-mediated T cell signaling, activates a T cell response that is approximately 2-fold greater than that of a patient-derived PRAME-specific TCR (e.g., activates a T cell response that is approximately 2-fold greater than, or approximately 3-fold greater than, or approximately 4-fold greater than that of a patient-derived PRAME-specific TCR).

[0140] The TCRs of the present invention can have one or more of the above biological characteristics, or any combination thereof. Other biological characteristics of the antigen-binding proteins of the present invention will be apparent to those skilled in the art from the present 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 polynucleotide encoding a protein can be inserted by covalent attachment such that it results in the expression of the protein and / or the cloning of the polynucleotide. Such vectors may also be referred to as "expression vectors." The isolated polynucleotide can be inserted into the vector using any suitable method known in the art. For example, without limitation, the vector can be digested using appropriate restriction enzymes and then ligated to the isolated polynucleotide having matching restriction ends. An expression vector has the ability to incorporate and express a heterologous or modified nucleic acid sequence encoding at least a portion of a gene product that can be transcribed intracellularly. In most cases, the RNA molecule is then translated into a protein. An expression vector can contain various control sequences, which refer to nucleic acid sequences necessary for the transcription or translation of an operably linked coding sequence in a particular host organism. In addition to the control sequences that regulate transcription and translation, vectors and expression vectors can further contain nucleic acid sequences that perform other functions, which are discussed below. Expression vectors can contain additional elements. For example, an expression vector can have two replication systems and thus can be maintained in two organisms, for example, human cells for expression and a prokaryotic host for cloning and amplification.

[0142] Expression vectors can 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 the 3'UTR AAUAAA transcription termination sequence for efficient gene transcription and translation in their respective host cells. Other suitable promoters include constitutive promoters of simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV immediate-early promoter, and Rous sarcoma virus promoter. Human gene promoters can also be used, including but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In certain embodiments, inducible promoters are also contemplated as part of vectors expressing chimeric antigen receptors. This provides a molecular switch that can turn on or turn off the expression of the polynucleotide sequence of interest. Examples of inducible promoters include but are not limited to metallothionein promoter, glucocorticoid promoter, progesterone promoter, or tetracycline promoter.

[0143] Expression vectors can have additional sequences such as 6× histidine (SEQ ID NO: 954), c-Myc, and FLAG tags incorporated into the expressed TCR. Thus, expression vectors can be engineered to include 5' and 3' untranslated regulatory sequences, which can sometimes function as enhancer sequences, promoter regions, and / or terminator sequences that can facilitate or enhance the efficient transcription of the nucleic acid of interest carried by the expression vector. Expression vectors can 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 can include selectable markers for maintenance of the vector in the host or recipient cell.

[0144] Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, but are not limited to, bacteriophages such as lambda phage or M13 phage, and animal viruses such as plasmids, phagemids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs) which are artificial chromosomes. Examples of categories of animal viruses 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 are the Lenti-X (trademark) bicistronic expression system (Neo) vector (Clontrch), pClneo vector (Promega) for expression in mammalian cells, pLenti4 / V5-DEST (trademark), pLenti6 / V5-DEST (trademark), and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequences of the TCRs disclosed herein can be ligated to such expression vectors for the expression of chimeric proteins in mammalian cells.

[0145] In certain embodiments, the nucleic acid encoding the TCR of the invention is provided in a viral vector. The viral vector can be derived from a retrovirus, a lentivirus, or a foamy virus. As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into viral vector particles. The viral vector can contain the coding sequences of various proteins described herein in place of non-essential viral genes. The vector and / or particle can be utilized for the purpose of transferring DNA, RNA, or other nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0146] In certain embodiments, the viral vector comprising the TCR coding sequence described herein is a retroviral vector or a lentiviral vector. The term "retroviral vector" refers to a vector that contains structural and functional genetic elements primarily derived from retroviruses. The term "lentiviral vector" refers to a vector that contains structural and functional genetic elements outside of the LTRs, primarily derived from lentiviruses.

[0147] Retroviral vectors for use herein can be derived from any known retrovirus (e.g., c-type retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV)). The "retroviruses" of the invention also include lentiviruses of the Retroviridae family such as human T-cell leukemia virus, HTLV-1 and HTLV-2, and 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] The lentiviral vector for use in this specification refers to a vector derived from lentiviruses, which are a group (or genus) of retroviruses that cause slowly developing diseases. Viruses included in this group are HIV (human immunodeficiency virus including HIV type 1 and HIV type 2), Visna / maedi, caprine arthritis-encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). Preparation of recombinant lentiviruses can be achieved using the methods by Dull et al. and Zufferey et al. (Dull et al., J. Virol., 1998; 72:8463-8471 and Zufferey et al., J. Virol. 1998; 72:9873-9880).

[0149] Retroviral vectors (i.e., both lentiviral and non-lentiviral) for use in the present invention can be generated using standard cloning techniques by combining the desired DNA sequences according to the instructions and adaptations described herein (Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals, Eglitis, et al. (1985) Science 230:1395-1398, Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464, Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018, Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145, Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043, Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381, Chowdhury et al. (1991) Science 254:1802-1805, van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644, Kay et al. (1992) Human Gene Therapy 3:641-647, Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895, Hwu et al. (1993) J. Immunol 150:4104-4115, U.S. Patent No. 4,868,116, U.S. Patent No. 4,980,286, PCT Application WO89 / 07136, PCT Application WO89 / 02468, PCT Application WO89 / 05345, and PCT Application WO92 / 07573).

[0150] Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) arrays for use in generating vectors include, for example, genomic RNA and cDNA available from commercial sources including the Type Culture Collection (ATCC), Rockville, Md. The sequences can also be chemically synthesized.

[0151] In the expression of a PRAME TCR, a vector can be introduced into a host cell to enable the expression of the polypeptide within the host cell. The expression vector can contain various elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selectable marker, and a signal sequence. These elements can be appropriately selected by those skilled in the art as described above. For example, the promoter sequence can be selected to promote transcription of the polynucleotide in the vector. Suitable promoter sequences include, but are not limited to, the T7 promoter, the T3 promoter, the SP6 promoter, the β-actin promoter, the EF1a promoter, the CMV promoter, and the SV40 promoter. The enhancer sequence can be selected to enhance transcription of the polynucleotide. The selectable marker can enable selection of host cells into which the vector has been inserted from those into which it has not been inserted. For example, the selectable marker can be a gene conferring antibiotic resistance. The signal sequence can be selected to enable the expressed polypeptide to be transported outside the host cell.

[0152] In the cloning of a polynucleotide, a vector is introduced into a host cell (an isolated host cell) to enable replication of the vector itself, thereby amplifying copies of the polynucleotide contained therein. Cloning vectors generally can contain, but are not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements can be appropriately selected by those skilled in the art. For example, the origin of replication can be selected to promote autonomous replication of the vector in the host cell.

[0153] In certain embodiments, the present disclosure provides an isolated host cell comprising the vectors provided herein. A host cell comprising a vector can be useful in the expression or cloning of a polynucleotide contained in the vector. Suitable host cells can 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 can include, but are not limited to, Gram-negative or Gram-positive organisms, such as enterobacteria, for example, Escherichia coli, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Serratia, for example Serratia marcescans, and Shigella, as well as bacilli such as B. subtilis and B. licheniformis, Pseudomonas aeruginosa such as P. aeruginosa, and Streptomyces.

[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 can be integrated into the host cell DNA or maintained extrachromosomally. The nucleic acid can be maintained transiently or represent stable introduction. Transfection can be accomplished by a variety of means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and gene gun, but is not limited thereto. Transduction refers to the delivery of genes using viral vectors or retroviral vectors, rather by means of viral infection rather than transfection. In certain embodiments, the retroviral vector is transduced by packaging the vector into virions prior to contact with the cell. For example, the nucleic acid encoding the PRAME TCR of the present invention carried by a retroviral vector can be transduced into cells through infection and proviral integration.

[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 total genetic material within a cell. The terms "genetically modified cell", "modified cell", and "redirected cell" are used interchangeably.

[0156] In particular, the TCR of the present invention is introduced into immune effector cells and expressed, thereby redirecting their specificity to a target antigen of interest (e.g., HLA-A2-presented PRAME peptide, e.g., amino acid residues 312-320 or 425-433 of PRAME).

[0157] The present invention provides a method for generating immune effector cells that express a TCR as described herein. In some embodiments, the method comprises transfecting or transducing immune effector cells (e.g., immune effector cells isolated from a subject such as a subject having a PRAME-related disease or disorder) such that the immune effector cells express one or more TCRs as described herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further in vitro manipulation. Such cells can then be re-administered directly to the individual. In further embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro and then genetically modified to express a TCR. In this regard, the immune effector cells can be cultured either before or after being genetically modified (i.e., transduced or transfected to express a TCR as described herein).

[0158] Prior to in vitro manipulation or genetic modification of the immune effector cells described herein, the cell source can be obtained from a subject. In particular, immune effector cells for use with the TCRs described herein include T cells.

[0159] T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymic effluent, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art, such as FICOLL separation. In some embodiments, cells from an individual's circulating blood are obtained by apheresis. Apheresis products typically contain lymphocytes, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets, including T cells. In some embodiments, the cells recovered by apheresis can be washed to remove the plasma fraction and to place the cells in an appropriate buffer or medium for subsequent processing. In some embodiments of the invention, the cells are washed with PBS. In alternative embodiments, the wash solution can be calcium-free and magnesium-free or lacking in many, but not all, divalent cations. As will be recognized by those of skill in the art, the wash step can be accomplished by methods known to those of skill in the art, such as by using a semi-automatic flow-through centrifuge. After washing, the cells can be resuspended in a variety of biocompatible buffers or other physiological saline solutions, with or without buffer. In certain embodiments, unwanted 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 on 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 a T cell population by negative selection can be achieved by a combination of antibodies against surface markers specific to the 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 the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate the cell population of interest for use in the present invention.

[0161] PBMCs can be used directly for TCR-based genetic modification using the methods described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated and, in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion.

[0162] Can immune effector cells, such as T cells, be genetically modified after isolation using known methods, or can immune effector cells be activated and expanded in vitro (or differentiated in the case of progenitor cells) before being genetically modified? In another embodiment, immune effector cells, such as T cells, are genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector containing a nucleic acid encoding a TCR) and then activated and expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Patent No. 6,905,874, U.S. Patent No. 6,867,041, U.S. Patent No. 6,797,514, WO2012 / 079000, US2016 / 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 a PRAME TCR disclosed herein.

[0164] TCR-expressing immune effector cells prepared as described herein can be utilized in methods and compositions for adoptive immunotherapy according to known techniques or modifications thereof that will be apparent to those skilled in the art based on the present 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] III. Pharmaceutical Compositions The present invention provides a therapeutic composition comprising the PRAME TCR of the present invention, or immune effector cells comprising the PRAME TCR of the present invention. The therapeutic composition according to the present invention is administered together with a suitable carrier, an excipient, and other agents incorporated into the formulation to provide improved delivery, delivery, resistance, etc. Many suitable formulations can be found in Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, a formulary 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 glycols of various molecular weights), semi-solid gels, and carbowax-containing semi-solid mixtures. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

[0166] Depending on the severity of the condition, the frequency and duration of treatment can be adjusted.

[0167] In certain embodiments, after the initial dose, subsequent doses of the PRAME TCR of the present invention or immune effector cells comprising the PRAME TCR of the present invention may be administered in an amount approximately the same as or less than the initial dose.

[0168] In certain situations, the pharmaceutical composition can be delivered by a sustained release system. In some embodiments, a pump can be used.

[0169] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal, and intramuscular injections, infusions, etc. The TCRs, pharmaceutical compositions, and cells described herein can be administered via parenteral administration. The preparations of the present disclosure can be prepared by methods known in the art. For example, the preparation can be prepared by dissolving, suspending, or emulsifying the above antigen-binding protein or a salt thereof in a sterile aqueous medium or an oily medium conventionally used for injection. Examples of the aqueous medium for injection include physiological saline, isotonic solutions containing glucose, and other adjuvants, which can be used in combination with appropriate solubilizing agents 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)]. As the oily medium, for example, sesame oil, soybean oil, etc. are used, and they can be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. The injection thus prepared is preferably filled into appropriate ampoules.

[0170] In some embodiments, TCR-expressing immune effector cells are first harvested from their medium and then formulated by washing and concentrating the cells in a therapeutically effective amount in a medium and container system suitable for administration (a “pharmaceutically acceptable” carrier). Suitable infusion media can be any isotonic media formulation, typically physiological saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), but 5% dextrose in water or lactated Ringer's solution can also be utilized. The infusion medium can be supplemented with human serum albumin.

[0171] The therapeutically effective number of cells in the composition is typically greater than 10 2 cells and up to 10 6 cells, including 10 8 cells or 10 9 cells, and 10 10It can be more than one. The number of cells depends on the ultimate use for which the composition is intended and the cell type contained therein.

[0172] The cells can be autologous or heterologous to the patient receiving the therapy. If desired, the treatment can also include administration of mitogens (e.g., PHA), 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 immune effector cell population of the present invention can 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 can comprise a TCR-expressing immune effector cell population such as the T cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose or dextran, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0174] IV. Therapeutic Use of Immune Effector Cells Comprising PRAME TCR or PRAME TCR The anti-tumor immune response induced in a subject by administering a TCR that expresses the T cells described herein using the methods described herein, or other methods known in the art, can include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses that can kill infected cells. A humoral immune response primarily mediated by helper T cells that can activate B cells and thus result in antibody production can also be induced. A variety of techniques can be used to analyze the type of immune response induced by the compositions of the invention, and they are well described in the art, for example, Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, N.Y.

[0175] Therefore, the PRAME TCR of the present invention is useful, inter alia, for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by PRAME. For example, the present invention provides a method (inhibiting tumor growth) for treating a PRAME-related disease or disorder such as a PRAME-related cancer (e.g., a PRAME-positive cancer), the method comprising administering to a patient in need of such treatment a PRAME TCR (or a pharmaceutical composition comprising the PRAME TCR or a plurality of cells comprising the PRAME TCR) as described herein, and provides a PRAME TCR (or a pharmaceutical composition comprising the PRAME TCR) for use in the treatment of a PRAME-related cancer. The antigen-binding protein of the present invention is useful for the treatment, prevention, and / or amelioration of a disease or disorder or condition such as a PRAME-related cancer, and / or for the amelioration of at least one symptom associated with such disease, disorder, or condition. In the context of the treatment methods described herein, the PRAME TCR (or pharmaceutical composition or plurality of cells) can be administered as a monotherapy (i.e., as the sole therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0176] Accordingly, the present invention provides a method for treating an individual diagnosed with or suspected of having or at risk of developing a PRAME-related disease or disorder (e.g., a PRAME-related cancer), the method comprising administering to the individual a therapeutically effective amount of a TCR-expressing immune effector cell as described herein.

[0177] In one embodiment, the present invention provides a method for treating a subject diagnosed with PRAME-positive cancer, the method comprising removing immune effector cells from a subject diagnosed with PRAME-positive cancer; genetically modifying the immune effector cells using a vector comprising a nucleic acid encoding a TCR of the present invention, thereby producing a modified immune effector cell population; and administering the modified immune effector cell population to the subject. In some embodiments, the immune effector cells comprise T cells.

[0178] The methods of administering the cell compositions described herein include any method effective to result in the reintroduction of in vitro genetically modified immune effector cells, which cells either directly express the TCR of the present invention in the subject or express the TCR upon reintroduction of genetically modified progenitor cells of immune effector cells that differentiate into mature immune effector cells upon introduction into the subject. One method includes transducing peripheral blood T cells in vitro with a nucleic acid construct according to the present invention and returning the transduced cells to the 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 cancer, prostate cancer, breast cancer, astrocytic tumors, glioblastoma multiforme, anaplastic astrocytoma, brain tumors, fallopian tube cancer, primary peritoneal cancer, advanced solid tumors, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin disease, multiple myeloma, metastatic solid tumors, colorectal cancer, stomach cancer, gastric cancer, rhabdomyosarcoma, myxoid round cell liposarcoma, or recurrent non-small cell lung cancer. In some embodiments, PRAME-related cancers are 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 carcinoma of the corpus uteri, carcinosarcoma of the uterus, testicular germ cell tumor, uveal melanoma, papillary renal cell carcinoma of the kidney, clear cell renal cell carcinoma of the kidney, thymoma, colon adenocarcinoma, squamous cell carcinoma of the cervix, cervical tumors, pancreatic adenocarcinoma, liver cancer, hepatocellular carcinoma, mesothelioma, or myxoid round cell liposarcoma.

[0180] The TCR can be used to treat the initial or late symptoms of PRAME-related cancers. In some embodiments, the TCR of the present invention can be used to treat progressive or metastatic cancers. The TCR is useful for reducing, inhibiting, or shrinking tumor growth. In certain embodiments, treatment with the TCR of the present invention results in a regression of more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the tumors in the subject. In certain embodiments, the TCR can be used to prevent tumor recurrence. In certain embodiments, the TCR is useful for extending the progression-free survival or overall survival in a subject having a PRAME-related cancer. In some embodiments, the TCR is useful for reducing the toxicity resulting from chemotherapy or radiotherapy while maintaining long-term survival in a patient suffering from a PRAME-related cancer.

[0181] One or more TCRs of the present invention can 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 contemplated herein to prophylactically use one or more TCRs of the present invention in a patient at risk of developing a disease or disorder such as a 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 a pharmaceutical composition for treating a patient suffering from a PRAME-related disease or disorder such as PRAME-related cancer. In another embodiment of the present invention, the TCR is used as adjuvant therapy with any other agent or any other therapy known to those skilled in the art useful for the treatment of PRAME-related cancer.

[0184] Combination therapy can include the PRAME TCR of the present invention (e.g., immune effector cells comprising the TCR of the present invention), or the pharmaceutical composition of the present invention, and any additional therapeutic agent that can be advantageously combined with the TCR of the present invention. The TCR of the present invention can be synergistically combined with one or more anti-cancer agents or therapies used to treat or inhibit PRAME-related diseases or disorders such as PRAME-positive cancers, e.g., liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumors, glioblastoma multiforme, anaplastic astrocytoma, brain tumors, fallopian tube cancer, primary peritoneal cancer, advanced solid tumors, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin 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] As used herein, it is contemplated to use the TCR of the present invention in combination with immunostimulatory therapy and / or immunomodulatory therapy 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 "removing the brakes" or "stepping on the accelerator" of suppressed immune cells. Examples include targeting other checkpoint receptors, vaccination, and adjuvants. Immunomodulatory modalities can 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 invention are a PD-1 inhibitor (e.g., an anti-PD-1 antibody such as nivolumab, pembrolizumab, pidilizumab, BGB-A317, or REGN2810), a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody such as avelumab, atezolizumab, durvalumab, MDX-1105, or REGN3504), a CTLA-4 inhibitor (e.g., ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T cell coinhibitor or ligand (e.g., an antibody against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist [e.g., a "VEGF-Trap" such as aflibercept or other VEGF inhibitory fusion proteins shown in US7,087,411, or an anti-VEGF antibody or antigen-binding fragment thereof (e.g., bevacizumab or ranibizumab), or a small molecule kinase inhibitor of the VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib)], an Ang2 inhibitor (e.g., nesvacumab), a transforming growth factor β (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor (e.g., erlotinib, cetuximab), an NY-ESO-1 inhibitor (e.g., an anti-NY-ESO-1 antibody), a CD20 inhibitor (e.g., an anti-CD20 antibody such as rituximab), an antibody against a tumor-specific antigen [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], a vaccine (e.g., BacillusBacillus Calmette-Guérin (BCG), cancer vaccines, adjuvants that increase antigen presentation (e.g., granulocyte macrophage colony-stimulating factor), co-stimulatory agents, bispecific antibodies (e.g., CD3×CD20 bispecific antibodies, PSMA×CD3 bispecific antibodies, or bispecific antibodies that bind to tumor antigens and have co-stimulatory activity, such as bispecific antibodies that act as co-stimulatory agents), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, 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, non-steroidal anti-inflammatory drugs), dietary supplements (e.g., antioxidants), or any other therapy for treating cancer can be used in combination. In certain embodiments, the TCRs of the present invention can be used in combination with cancer vaccines, including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, etc., to enhance the anti-tumor response.

[0187] Examples of cancer vaccines that can be used in combination with the TCRs of the present invention include MAGE3 vaccines for melanoma and bladder cancer, MUC1 vaccines for breast cancer, EGFRv3 (e.g., linderpepimut) for brain tumors (including glioblastoma multiforme), ALVAC-CEA (for CEA+ cancers), and NY-ESO-1 vaccines (e.g., for melanoma).

[0188] In certain embodiments, the PRAME TCR of the present invention can be administered in combination with radiation therapy in a method of generating a long-term persistent anti-tumor response and / or enhancing the survival rate of a patient having cancer. In some embodiments, the PRAME TCR of the present invention can be administered before, simultaneously with, or after administering radiation therapy to a cancer patient. For example, radiation therapy can administer the PRAME TCR of the present invention to a tumor lesion in one or more doses, and then one or more additional doses can be administered. In some embodiments, radiation therapy is locally administered to a tumor lesion to enhance the local immunogenicity of the patient's tumor (therapeutic radiation) and / or kill tumor cells (ablative radiation), and then the PRAME TCR of the present invention can be administered systemically.

[0189] Additional therapeutic agents / components can be administered before, simultaneously with, or after administration of the PRAME TCR of the present invention. For purposes of this disclosure, such dosing regimens are considered to be an administration of the PRAME TCR "in combination with" a second therapeutic active ingredient.

[0190] An additional therapeutic active ingredient can be administered to a subject prior to administration of the PRAME TCR of the present invention. In other embodiments, the additional therapeutic active ingredient can be administered to a subject after administration of the PRAME TCR of the present invention. In still other embodiments, the additional therapeutic active ingredient can be administered to a subject simultaneously with the administration of the PRAME TCR of the present invention. "Simultaneous" administration, for the purposes of the present invention, includes, for example, administering the PRAME TCR and the additional therapeutic active ingredient to a subject in a single dosage form (e.g., co-formulation), or administering to the subject in separate dosage forms (administered within about 30 minutes of each other). When administered in separate dosage forms, each dosage form can be administered via the same route, or each dosage form can 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, for the purposes of this disclosure, to be "simultaneous administration". For the purposes of this disclosure, administration of the PRAME TCR "before", "simultaneously with", or "after" (as these terms are defined above) administration of the additional therapeutic active ingredient is considered to be administration of the PRAME TCR "in combination with" the additional therapeutic active ingredient.

[0191] The present invention is further illustrated by the following examples, which are not intended to be limiting in any way. The entire contents of all references, patents, and published patent applications cited throughout this application are hereby incorporated by reference into this specification.

Examples

[0192] Example 1. Identification of PRAME-Specific T Cell Receptors A VelociT™ mouse, a humanized mouse for components of the cellular immune system (see, for example, PCT Publication No. WO2016 / 164492, the entire contents of which are incorporated herein by reference), was immunized with either the PRAME (312-320) peptide (RLDQLLRHV; SEQ ID NO: 929) or the PRAME (425-433) peptide (SLLQHLIGL; SEQ ID NO: 930) specifically presented by human HLA-A2, diluted in PBS, and mixed in equal amounts with an adjuvant (e.g., complete Freund's adjuvant (CFA; Chondrex, Inc.)). Spleen suspensions from mice immunized with the wither peptide were obtained separately and dissociated. Red blood cells were lysed in ACK lysis buffer (Life Technologies), and splenocytes were suspended in RPMI complete medium. The isolated splenocytes were sorted, and single T cells that bind to the PRAME peptide (either PRAME (312-320) or PRAME (425-433)) used for immunization in the context of MHC were isolated by fluorescence-activated cell sorting (FACS). The isolated T cells were seeded into single-well plates and mixed with TCR alpha and beta variable region-specific PCR primers. cDNA in each single T cell was synthesized via a reverse transcriptase (RT) reaction. Next, each of the resulting RT products was divided and transferred to two corresponding wells for subsequent TCR beta and alpha PCR. One set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific for the TCR beta variable region leader sequence, or a 5' degenerate primer specific for the TCR alpha chain variable region leader sequence, and a 3' primer specific for the TCR constant region to generate an amplicon. Next, it was amplified again by PCR using a 5' degenerate primer specific for the TCR beta variable region framework 1, or a 5' degenerate primer specific for the TCR alpha chain variable region framework 1, and a 3' primer specific for the TCR constant region to generate an amplicon for cloning. The PCR products derived from TCR beta and alpha were cloned into expression vectors containing the beta constant region and the alpha constant region, respectively.An expression vector expressing a full-length beta chain and an alpha chain pair was transfected into CHO cells and tested for binding to a commercially available PRAME / HLA tetramer reagent. CHO cells were incubated with soluble HLA-A2:PRAME(312 - 320) or HLA:A2:PRAME(425 - 433) (MBL International, Woburn, MA) tetramers and an antibody specific for the mouse TCR constant region (clone H57-597) (Biolegend, San Diego, CA). Samples were then analyzed on an LSR Fortessa X-20 (BD Biosciences, San Jose, CA). To calculate the percentage of tetramer-positive cells, FlowJo (LLC, Ashland, OR) was used to set antigen-positive (Ag+) gating based on a negative control TCR that did not bind to the HLA-A2:PRAME(312 - 320) or HLA:A2:PRAME(425 - 433) (MBL International, Woburn, MA) tetramers. All Ag+ TCRs had a FlowJo criterion of ≥1% of cells in Ag+ gating with a 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 frequency of cells, 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 frequency of cells, i.e., the percentage (%) of tetramer-positive cells in Ag+ gating, represents the TCRs shown in the first column of Table 2.

[0193] A detailed list of the CDR1, CDR2, and CDR3 amino acid sequences of the beta-chain variable domain of the TCR specified by PRAME (312 - 320) as described above, and the CDR1, CDR2, and CDR3 amino acid sequences of the alpha-chain variable domain, is provided in Table 3. A detailed list of the CDR1, CDR2, and CDR3 polynucleotide sequences of the beta-chain variable domain of the TCR specified by PRAME (312 - 320) as described above, and the CDR1, CDR2, and CDR3 polynucleotide sequences of the alpha-chain variable domain, is provided in Table 4. Table 5 provides the amino acid sequences and nucleotide sequences of the beta-chain variable region and alpha-chain variable region of the TCR specified by PRAME (312 - 320).

[0194] A detailed list of the CDR1, CDR2, and CDR3 amino acid sequences of the beta-chain variable domain of the TCR specified by PRAME (425 - 433) as described above, and the CDR1, CDR2, and CDR3 amino acid sequences of the alpha-chain variable domain, is provided in Table 6. A detailed list of the CDR1, CDR2, and CDR3 polynucleotide sequences of the beta-chain variable domain of the TCR specified by PRAME (425 - 433) as described above, and the CDR1, CDR2, and CDR3 polynucleotide sequences of the alpha-chain variable domain, is provided in Table 7. Table 8 provides the amino acid sequences and nucleotide sequences of the beta-chain variable region and alpha-chain variable region of the TCR specified by PRAME (425 - 433).

[0195] Table 9 provides the TCR gene families of the alpha variable region, beta variable region, and binding region of the isolated TCR specified by PRAME (312 - 320), and Table 11 provides the amino acid sequence identifiers and polynucleotide sequence identifiers of the alpha variable chain, beta variable chain, and CDRs of the TCR specified by PRAME (312 - 320).

[0196] Table 10 provides the TCR gene families of the isolated TCR alpha variable region, beta variable region, and binding region identified by PRAME (425 - 433), and Table 12 provides the amino acid sequence identifiers and polynucleotide sequence identifiers of the TCR alpha variable chain, beta variable chain, and CDRs of the TCR 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 Gene disruption was used to generate Jurkat cell lines lacking expression of endogenous TCRα and TCRβ. These cells were then engineered at genomic landing pad sites that allow for single-copy Cre recombinase-mediated insertion of transgenic TCR constructs. An activator protein 1 (AP1) response element-driven luciferase reporter was then incorporated into this parental bioassay cell line. Specific TCR bioassay strains were generated by Cre-mediated insertion of constructs expressing TCRα and TCRβ sequences from VelociT®.

[0198] Jurkat bioassay stocks expressing the TCR constructs were sorted and made homogeneous using fluorescence-activated cell sorting (FACS) and then tested in a peptide-MHC stimulation assay. HEK293T cells (HLA-A2*01) were seeded into assay wells containing various dilutions of the antigenic PRAME peptide (PRAME425-433; SEQ ID NO: 930) or an irrelevant HLA-A2-restricted peptide (SLLMWITQC; SEQ ID NO: 953). These dilutions were prepared as shown in Table 13.

Table 13

[0199] After a 2-hour incubation, the engineered Jurkat cells were added to the wells at a Jurkat cell:293T cell ratio of 3:1 and incubated for an additional 5 hours. The activity of the luciferase reporter was determined by measuring the endpoint luminescence output in the assay wells. The PRAME-specific TCR mediated dose-dependent activation of the AP1 reporter in response to HLA-A2*01 HEK293T cells pulsed with the cognate peptide, but this was not seen in cells pulsed with the irrelevant peptide. EC50 data are shown in Table 14 below.

Table 14-1

Table 14-2

[0200] Equivalents One of ordinary skill in the art can recognize or confirm many equivalents to the specific embodiments of the invention described herein using only routine experimentation, and such equivalents are intended to be encompassed by the following claims. The contents of all references, patents, and published patent applications cited throughout this application are hereby incorporated by reference into this specification.

Claims

1. A T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, said TCR comprising alpha chain variable domain complementarity determining regions (CDRs) 1, CDR2 and CDR3, and beta chain variable domain CDR1, CDR2 and CDR3, contained within the TCR alpha chain variable domain / TCR beta chain variable domain amino acid sequence pair of SEQ ID NO:789 / 791.

2. The TCR described in claim 1, wherein the alpha chain variable domains CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 319, 320 and 321, respectively, and the beta chain variable domains CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 322, 323 and 324, respectively.

3. A TCR described in claim 1 or 2, wherein the TCR alpha chain variable domain / TCR beta chain variable domain comprises the amino acid sequence pair of sequence numbers 789 / 791.

4. A TCR described in any one of claims 1 to 3, further comprising a detectable moiety.

5. A pharmaceutical composition comprising the TCR described in any one of claims 1 to 4 and a pharma- ceutically acceptable carrier or diluent.

6. A cell presenting a TCR described in any one of claims 1 to 4.

7. A polynucleotide molecule comprising a polynucleotide sequence encoding the alpha chain variable domain of a TCR described in any one of claims 1 to 4.

8. The polynucleotide molecule described in claim 7, comprising a nucleotide sequence of SEQ ID NO:

790.

9. A polynucleotide molecule comprising a polynucleotide sequence encoding the beta chain variable domain of a TCR described in any one of claims 1 to 4.

10. The polynucleotide molecule described in claim 8, comprising the nucleotide sequence of SEQ ID NO:

792.

11. a) a polynucleotide molecule according to any one of claims 7 to 10; or b) a polynucleotide molecule according to claim 7 or 8, and a polynucleotide molecule according to claim 9 or 10. A vector comprising:

12. A cell comprising a polynucleotide described in any one of claims 7 to 10, or a vector described in claim 11.

13. A pharmaceutical composition comprising a plurality of cells described in claim 12 and a pharma- ceutically acceptable carrier or diluent.

14. A composition comprising a TCR according to any one of claims 1 to 4, or a plurality of cells according to claim 6, or a pharmaceutical composition comprising a TCR according to claim 5, or a plurality of isolated cells according to claim 13, for treating a subject having a PRAME-associated disease or disorder.

15. The composition described in claim 14, wherein the PRAME-associated disease or disorder is a PRAME-associated cancer.

16. The PRAME-associated cancer is liposarcoma, neuroblastoma, myeloma, melanoma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, esophageal squamous cell carcinoma, hepatocellular cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, ovarian epithelial cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Hodgkin's disease, multiple myeloma, metastatic solid tumor, colorectal cancer, stomach cancer, gastric cancer, 16. The composition of claim 15, wherein the cancer is selected from the group consisting of rhabdomyosarcoma, myxoid round cell liposarcoma, uterine 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, and recurrent non-small cell lung cancer.

17. A composition described in any one of claims 14 to 16, characterized in that it is administered to the subject in combination with a second therapeutic agent.

18. The composition described in any one of claims 14 to 17, characterized in that it is administered to the subject subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.

19. A polynucleotide molecule encoding a T cell receptor (TCR) that specifically binds to an antigen (PRAME) peptide preferentially expressed in HLA-A2-presenting melanoma, said TCR comprising alpha chain variable domain complementarity determining regions (CDRs) 1, CDR2 and CDR3, and beta chain variable domain CDR1, CDR2 and CDR3, contained within the TCR alpha chain variable domain / TCR beta chain variable domain amino acid sequence pair of SEQ ID NO:789 / 791.

20. The polynucleotide molecule of claim 19, wherein the alpha chain variable domains CDR1, CDR2 and CDR3 are encoded by the nucleotide sequences of SEQ ID NOs: 559, 560 and 561, respectively, and the beta chain variable domains CDR1, CDR2 and CDR3 are encoded by the nucleotide sequences of SEQ ID NOs: 562, 563 and 564, respectively.

21. A polynucleotide molecule described in claim 19 or 20, wherein the TCR alpha chain variable domain / TCR beta chain variable domain is encoded by the nucleotide sequence pair of SEQ ID NO:790 / 792.

22. A vector comprising a polynucleotide molecule described in any one of claims 19 to 21.

23. A cell containing the vector described in claim 22.

24. A composition comprising a plurality of cells according to claim 23 for treating a subject having a PRAME-associated disease or disorder.

25. The composition described in claim 24, wherein the PRAME-associated disease or disorder is a PRAME-associated cancer.

26. The composition described in claim 24 or 25, characterized in that the TCR and the multiple cells are administered to the subject in combination with a second therapeutic agent.

Images