Novel tumor antigens and their use against melanoma

Abstract

While immune checkpoint block (ICB) therapy has significantly improved outcomes for metastatic melanoma, most patients do not achieve long-term benefits. Previous studies have suggested that treatment failure is partly due to insufficient immune recognition of tumor antigens (TAs). Cancer vaccines may potentially offer a complementary approach to enhance anti-tumor immunity and act synergistically with ICIs. Novel tumor antigens shared by the majority of melanoma cells are described herein. Some of the tumor antigens described herein are derived from abnormally expressed non-mutant genomic sequences, such as intronic and intergenetic sequences, which are not expressed in normal tissues. Nucleic acids, compositions, cells, and vaccines derived from these tumor antigens are described. The use of tumor antigens, nucleic acids, compositions, cells, and vaccines for the treatment of melanoma is also described.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 496,084, filed on 14 April 2023, and U.S. Provisional Patent Application No. 63 / 515,147, filed on 24 July 2023, which are incorporated herein by reference in their entirety.

[0002] Sequence List The sequence listing was created on April 11, 2024, and is submitted with this specification as an XML file named G17971-00091_Seq Listing.xml, with a size of approximately 995,793 bytes. The contents of the aforementioned file are incorporated herein by reference in their entirety.

[0003] This invention generally relates to the field of cancer, and more specifically to the treatment of cancers such as melanoma. [Background technology]

[0004] Cutaneous melanoma is an invasive form of skin cancer with over 100,000 cases in the United States in 2021 (Siegel et al., CA Cancer J Clin. 2021;71(1):7-33), and it causes more than 7,000 deaths annually. Tumor staging is determined by histopathological and clinical factors, including Breslow depth of the tumor, ulceration status, spread of the disease from the primary tumor to lymph nodes, and the presence of metastasis. These factors are grouped into overall stages, indicated by Roman numerals from 0 to IV, with stage 0 being the earliest stage and stage IV being the most advanced. While patients within a given stage should theoretically have similar outcomes, significant heterogeneity still exists within each stage. For example, most patients with stage I disease are cured by surgery, but a significant minority (5-10%) may develop metastasis later.

[0005] While immune checkpoint blockade (ICB) therapy has significantly improved outcomes for metastatic melanoma, most patients do not experience long-term benefits. Previous studies have suggested that treatment failure is partly due to insufficient immune recognition of tumor antigens (TAs). In particular, immune targeting of TAs in melanoma has focused on TAs derived from non-synonymous genomic mutations.

[0006] Considering this, there is a need to identify tumor antigens that can induce a therapeutic immune response against melanoma. Such antigens can be used as vaccines (± immune checkpoint inhibitors) or as targets for T-cell receptor-based approaches (cell therapies, bispecific biologics).

[0007] This document references several sources, the contents of which are incorporated herein by reference in their entirety. [Overview of the Initiative]

[0008] In various aspects and embodiments, this disclosure provides the following items 1 to 79. 1. A tumor antigen peptide (TAP) that contains, or consists of, one of the amino acid sequences defined by any one of sequence numbers 1 to 505. 2. The TAP described in item 1, wherein the TAP is bound to an HLA-A*01:01 molecule and contains or consists of the sequence of SEQ ID NOs: 11, 14, 20, 27, 29, 37, 86, 87, 121, 150, 165, 173, 279, 288, 299, 308, 312, 316, 319, 331, 340, 352, 362, 363, 384, 395, 398, 411, 419, 429, 458, 474, 476, 487, 492, 500, or 501. 3. The TAP binds to the HLA-A*02:01 molecule, and is used in SEQ ID NOs: 1, 2, 4, 19, 22, 25, 40, 50, 52, 54, 60, 65, 67, 76, 81, 89, 91, 103, 107, 120, 124, 128, 135, 157, 175, 179, 186, 188, 201, 208, 218, 219, 228, 232, 233, 240, 250, A TAP as described in item 1, which includes or consists of the sequence 257, 261, 263, 265, 266, 282, 286, 298, 311, 315, 317, 325, 333, 337, 349, 367, 373, 387, 388, 393, 399, 404, 412, 414, 415, 422, 434, 437, 455, 480, 489, or 494. 4. The TAP described in item 1, wherein the TAP is bound to an HLA-A*02:09 molecule and contains or consists of the sequence of SEQ ID NOs: 22, 40, 50, 62, 83, 89, 232, 265, 282, 311, 315, 317, 337, 349, 367, 399, 407, 412, or 494. 5. The TAP described in item 1, wherein the TAP is bound to an HLA-A*03:01 molecule and contains or consists of the sequence of SEQ ID NOs. 209, 247, 272, 347, or 505. 6. The TAP described in item 1, wherein the TAP is bound to an HLA-A*23:01 molecule and contains or consists of the sequence of SEQ ID NOs: 48, 84, 129, 151, 152, 171, 174, 182, 223, 237, 245, 290, 358, 397, 426, 446, 481, 482, or 486. 7. The TAP described in item 1, wherein the TAP is bound to an HLA-A*24:02 molecule and comprises or consists of the sequence of SEQ ID NO: 31, 244, or 481. 8. The TAP described in item 1, wherein the TAP is bound to an HLA-A*25:01 molecule and contains or consists of the sequence of SEQ ID NOs: 11, 31, 249, 289, 403, 425, 464, or 490. 9. The TAP described in item 1, wherein the TAP is bound to an HLA-A*26:01 molecule and contains or consists of the sequence of SEQ ID NOs: 11, 20, 27, 39, 41, 42, 45, 114, 116, 122, 168, 181, 268, 284, 329, 364, 398, 417, 430, 440, 447, 452, 457, 460, 462, 475, 490, or 497. 10. The TAP described in item 1, wherein the TAP is bound to an HLA-A*30:01 molecule and contains or consists of the sequence of SEQ ID NOs. 26, 304, or 376. 11. The TAP described in item 1, wherein the TAP is bound to an HLA-A*30:02 molecule and contains or consists of the sequence of SEQ ID NOs: 117, 185, 190, 193, 197, 256, 294, 323, 471, or 485. 12. The TAP described in item 1, wherein the TAP is bound to an HLA-A*33:01 molecule and contains or consists of the sequence of sequence number 248. 13. The TAP binds to the HLA-A*68:01 molecule, and is present in SEQ ID NOs: 6-8, 12, 15, 23, 28, 41, 42, 47, 51, 53, 56, 59, 64, 66, 68, 70, 73-75, 77, 79, 80, 82, 85, 88, 92, 93, 96-99, 101, 102, 108-112, 118 , 119, 123, 130, 131, 134, 137, 138, 141, 142, 144~147, 149, 153, 154, 156, 158, 160, 161, 166, 169, 172, 176, 184, 198, 202, 203, 212, 214, 216, 217, 220, 224, 226, 229 ,234,236,239,248,254,269,270,275,280,283,287,293,300,301,303,306,310,314,320,328,332,348,354,355,359,360,366,369,370,372,381,382,386,389 TAP as described in item 1, which includes or consists of the sequence 390, 392, 394, 402, 405, 406, 409, 413, 418, 424, 428, 436, 441-445, 449, 456, 459, 465-468, 473, 479, 483, 488, 493, 499, or 502. 14. The TAP described in item 1, wherein the TAP is bound to an HLA-B*07:02 molecule and contains or consists of the sequence of SEQ ID NOs: 35, 36, 43, 104, 125, 127, 139, 140, 187, 191, 196, 206, 231, 243, 246, 251, 252, 260, 281, 291, 292, 321, 330, 361, 408, 432, 435, 439, 451, 461, 491, or 503. 15. The TAP described in item 1, wherein the TAP is bound to an HLA-B*08:01 molecule and contains or consists of the sequence of SEQ ID NOs. 21, 162, 189, 286, 307, 309, 317, 339, 343, 349, or 414. 16. The TAP described in item 1, wherein the TAP is bound to an HLA-B*13:02 molecule and comprises or consists of the sequence of SEQ ID NOs. 207, 311, 393, 400, or 455. 17. The TAP described in item 1, wherein the TAP is bound to an HLA-B*14:01 molecule and contains or consists of the sequence of sequence number 396. 18. The TAP described in item 1, wherein the TAP is bound to an HLA-B*15:01 molecule and contains or consists of the sequence of SEQ ID NOs: 11, 26, 30, 63, 113, 132, 148, 192, 195, 200, 207, 210, 222, 235, 238, 258, 262, 267, 276, 295, 297, 305, 309, 318, 326, 336, 342, 374, 377, 383, 401, 421, 427, 433, 438, 450, 463, 470, or 477. 19. The TAP described in item 1, wherein the TAP is bound to an HLA-B*18:01 molecule and contains or consists of the sequence of SEQ ID NOs: 3, 10, 33, 225, or 396. 20. The TAP described in item 1, wherein the TAP is bound to an HLA-B*37:01 molecule and contains or consists of the sequence of sequence number 302. 21. The TAP described in item 1, wherein the TAP is bound to an HLA-B*38:01 molecule and comprises or consists of the sequence of SEQ ID NOs. 26, 180, 338, 341, 378, 380, 416, 448, 454, 496, or 498. 22. The TAP described in item 1, wherein the TAP is bound to an HLA-B*40:01 molecule and contains or consists of the sequence of sequence number 215 or 322. 23. The TAP described in item 1, wherein the TAP is bound to an HLA-B*40:02 molecule and contains or consists of the sequence of sequence numbers 213, 215, 339, or 351. 24. The TAP described in item 1, wherein the TAP is bound to an HLA-B*44:02 molecule and comprises or consists of the sequence of SEQ ID NOs. 346, 353, 356, or 453. 25. The TAP described in item 1, wherein the TAP is bound to an HLA-B*49:01 molecule and contains or consists of the sequence of SEQ ID NOs: 9, 57, 78, 115, 136, 167, 255, 273, 274, 278, 313, 339, 480, 484, or 495. 26. The TAP described in item 1, wherein the TAP is bound to an HLA-B*51:01 molecule and contains or consists of the sequence of sequence number 227 or 259. 27. The TAP described in item 1, wherein the TAP is bound to an HLA-C*03:03 molecule and comprises or consists of the sequence of SEQ ID NOs. 24, 69, 204, or 388. 28. The TAP described in item 1, wherein the TAP is bound to an HLA-C*03:04 molecule and contains or consists of the sequence of SEQ ID NOs. 24, 49, 69, 159, 241, 242, or 334. 29. The TAP described in item 1, wherein the TAP is bound to an HLA-C*06:02 molecule and comprises or consists of the sequence of SEQ ID NOs: 13, 34, or 72. 30. The TAP described in item 1, wherein the TAP is bound to an HLA-C*07:01 molecule and contains or consists of the sequence of SEQ ID NOs: 13, 75, 133, 163, 183, 199, 205, 285, 334, 391, or 472. 31. The TAP described in item 1, wherein the TAP is bound to an HLA-C*07:02 molecule and comprises or consists of the sequence of SEQ ID NOs: 13, 32, 36, 105, 177, 339, 371, or 478. 32. The TAP described in item 1, wherein the TAP is bound to an HLA-C*08:02 molecule and comprises or consists of the sequence of SEQ ID NOs: 18, 54, or 379. 33. The TAP described in item 1, wherein the TAP is bound to an HLA-C*12:03 molecule and contains or consists of the sequence of SEQ ID NOs: 24, 49, 58, 71, 94, 162, 178, 242, 289, 344, 345, 357, 367, 368, 375, 423, or 431. 34. The TAP described in item 1, wherein the TAP is bound to an HLA-C*14:02 molecule and contains or consists of the sequence of SEQ ID NOs: 105, 271, 350, 365, 385, 410, 423, or 504. 35. A TAP described in any one of items 1-34, encoded by a sequence located in a non-protein-coding region of the genome or by a long non-coding RNA. 36. The non-protein-coding region of the genome is an inter-gene region, as described in item 35. 37. The TAP described in item 35, wherein the non-protein-coding region of the genome is an intron. 38. The TAP according to any one of items 1 to 37, wherein the TAP is conjugated to a molecule that increases the protease resistance, plasma protein binding, plasma half-life and / or intracellular osmosis of the TAP, such as a fatty acid, a protein (e.g., albumin), a sugar or polysaccharide, or a polymer (e.g., polyethylene glycol). 39. A combination that includes at least two of the TAPs defined in any one of items 1-38. 40. A synthetic long peptide (SLP) containing at least one of the amino acid sequences defined in item 1. 41. A nucleic acid that codes for one or more of the TAPs listed in items 1-38, the combinations listed in item 39, or the SLPs listed in item 40. 42. The nucleic acid according to item 41, wherein the nucleic acid is mRNA, and the mRNA optionally comprises one or more 5'-terminal modifications, 3'-terminal modifications, and / or modified nucleosides to increase the stability of the mRNA, improve translation, and / or reduce immunogenicity. 43. The nucleic acid described in item 41, wherein the nucleic acid is DNA. 44. The nucleic acid described in any one of items 41 to 43, wherein the nucleic acid is a component of a viral vector. 45. A vesicle or particle containing a TAP as described in any one of items 1-38, a combination as described in item 39, an SLP as described in item 40, or a nucleic acid as described in any one of items 41-44. 46. ​​The vesicle or particle according to item 45, wherein the vesicle is a lipid nanoparticle (LNP). 47. Vesicles or particles as described in item 45 or 46, containing cationic lipids. 48. A composition comprising a TAP as described in any one of items 1 to 38, a combination as described in item 39, an SLP as described in item 40, a nucleic acid as described in any one of items 41 to 44, or a vesicle or particle as described in any one of items 45 to 47, and a pharmaceutically acceptable carrier. 49. A vaccine comprising a TAP as described in any one of items 1 to 38, a combination as described in item 39, an SLP as described in item 40, a nucleic acid as described in any one of items 41 to 44, a vesicle or particle as described in any one of items 45 to 47, or a composition as described in claim 47, and an adjuvant. 50. An isolated major histocompatibility complex (MHC) class I molecule comprising a TAP described in any one of items 1 to 38 within its peptide bond groove. 51. An isolated MHC class I molecule described in item 50, in the form of a polymer. 52. The isolated MHC class I molecule described in item 51, wherein the polymer is a tetramer. 53. Isolated cells comprising (i) a TAP as described in any one of items 1 to 38, (ii) a combination as described in item 39, (iii) an SLP as described in item 40, (iv) a nucleic acid as described in any one of items 41 to 44, or (v) a vector containing a nucleotide sequence encoding a TAP as described in any one of items 1 to 38, a combination as described in item 39, or an SLP as described in item 40. 54. Isolated cells expressing a major histocompatibility complex (MHC) class I molecule containing a TAP described in any one of items 1-38 or a combination described in item 39 within a peptide binding groove on its surface. 55. Antigen-presenting cells (APCs), as described in item 53 or 54. 56. The cell described in item 55, wherein the APC is a dendritic cell. 57. A T cell receptor (TCR) or nucleic acid encoding the TCR, which specifically recognizes an isolated MHC class I molecule as described in any one of items 50-52, and / or an MHC class I molecule expressed on the surface of a cell as described in any one of items 54-56. 58. A soluble TCR, as described in item 57. 59. An antibody or an antigen-binding fragment thereof that specifically binds to an isolated MHC class I molecule as described in any one of items 50-52, and / or an MHC class I molecule expressed on the surface of a cell as described in any one of items 54-56, or a nucleic acid encoding the antibody or the antigen-binding fragment thereof. 60. A bispecific TCR, or a bispecific antibody, or its antigen-binding fragment, as described in item 57 or 58, or an antibody or its antigen-binding fragment as described in item 59. 61. The TCR, antibody or antigen-binding fragment described in item 60, wherein the bispecific antibody or its antigen-binding fragment is a single-stranded diabody (scDb). 62. The TCR, antibody, or antigen-binding fragment according to item 60 or 61, wherein the bispecific TCR, antibody, or antigen-binding fragment also specifically binds to a T cell signaling molecule. 63. The TCR, antibody or antigen-binding fragment described in item 62, wherein the T cell signaling molecule is a CD3 chain. 64. A chimeric antigen receptor (CAR), or a nucleic acid encoding the CAR, comprising an antibody or antigen-binding fragment thereof as described in item 59. 65. Isolated cells expressing the TCR described in item 57 or the CAR described in item 64 on their cell surface. 66. CD8 + Isolated cells, which are T lymphocytes, as described in item 65. 67. A cell population containing at least 0.5% isolated cells as defined in item 65 or 66. 68. A method for treating cancer in a subject, wherein an effective amount of the subject (a) A TAP containing or consisting of any one of the sequences defined in SEQ ID NOs: 1 to 505, or any combination thereof, or a synthetic long peptide (SLP) containing at least one of the sequences defined in SEQ ID NOs: 1 to 505, (b) A TAP as defined in (a), a combination thereof, or at least one nucleic acid encoding an SLP, (c) A vesicle or particle containing a TAP as defined in (a), a combination thereof, or an SLP, or at least one nucleic acid as defined in (b), (d) A composition comprising a TAP as defined in (a), a combination thereof, or an SLP, at least one nucleic acid as defined in (b), or a vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier. (e) A vaccine comprising a TAP as defined in (a), a combination thereof, or an SLP, at least one nucleic acid as defined in (b), a vesicle or particle as defined in (c), or a composition as defined in (d), and an adjuvant. (f) Cells that express major histocompatibility complex (MHC) class I molecules containing TAP or a combination thereof as defined in (a) within their peptide bond grooves on their surface, A cell expressing a T cell receptor (TCR) or chimeric antigen receptor (CAR) on its cell surface that specifically recognizes MHC class I molecules expressed on the surface of a cell as defined in (g)(f), or A method comprising administering a soluble TCR, antibody or its antigen-binding fragment, or CAR, or a nucleic acid encoding the soluble TCR, antibody, its antigen-binding fragment, or CAR, which specifically binds to an MHC class I molecule expressed on the surface of a cell as defined in (h)(f). 69. The method according to item 68, wherein the cancer is melanoma. 70. The method according to item 68 or 69, further comprising administering at least one additional antitumor agent or therapy to the subject. 71. The method according to item 70, wherein the at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiotherapy, or surgery. 72. For the manufacture of pharmaceuticals for the treatment of cancer in subjects, or for the manufacture of pharmaceuticals for the treatment of cancer in subjects, (a) A TAP containing or consisting of any one of the sequences defined in SEQ ID NOs: 1 to 505, or any combination thereof, or a synthetic long peptide (SLP) containing at least one of the sequences defined in SEQ ID NOs: 1 to 505, (b) A TAP as defined in (a), a combination thereof, or at least one nucleic acid encoding an SLP, (c) A vesicle or particle containing a TAP as defined in (a), a combination thereof, or an SLP, or at least one nucleic acid as defined in (b), (d) A composition comprising a TAP as defined in (a), a combination thereof, or an SLP, at least one nucleic acid as defined in (b), or a vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier. (e) A vaccine comprising a TAP as defined in (a), a combination thereof, or an SLP, at least one nucleic acid as defined in (b), a vesicle or particle as defined in (c), or a composition as defined in (d), and an adjuvant. (f) Cells that express major histocompatibility complex (MHC) class I molecules containing TAP or a combination thereof as defined in (a) within their peptide bond grooves on their surface, A cell expressing a T cell receptor (TCR) or chimeric antigen receptor (CAR) on its cell surface that specifically recognizes MHC class I molecules expressed on the surface of a cell as defined in (g)(f), or The use of a soluble TCR, an antibody or antigen-binding fragment thereof, or a CAR, or a nucleic acid encoding the soluble TCR, antibody, antigen-binding fragment thereof, or CAR, which specifically binds to an MHC class I molecule expressed on the surface of a cell as defined in (h)(f). 73. Use as described in item 72, provided the cancer is melanoma. 74. Uses described in item 72 or 73, further comprising the use of at least one additional antitumor agent or therapy. 75. The use described in item 74, wherein the at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiotherapy, or surgery. 76. A drug used to treat cancer in a subject, wherein the drug is (a) A TAP containing or consisting of any one of the sequences defined in SEQ ID NOs: 1 to 505, or any combination thereof, or a synthetic long peptide (SLP) containing at least one of the sequences defined in SEQ ID NOs: 1 to 505, (b) A TAP as defined in (a), a combination thereof, or at least one nucleic acid encoding an SLP, (c) A vesicle or particle containing a TAP as defined in (a), a combination thereof, or an SLP, or at least one nucleic acid as defined in (b), (d) A composition comprising a TAP as defined in (a), a combination thereof, or an SLP, at least one nucleic acid as defined in (b), or a vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier. (e) A vaccine comprising a TAP as defined in (a), a combination thereof, or an SLP, at least one nucleic acid as defined in (b), a vesicle or particle as defined in (c), or a composition as defined in (d), and an adjuvant. (f) Cells that express major histocompatibility complex (MHC) class I molecules containing TAP or a combination thereof as defined in (a) within their peptide bond grooves on their surface, A cell expressing a T cell receptor (TCR) or chimeric antigen receptor (CAR) on its cell surface that specifically recognizes MHC class I molecules expressed on the surface of a cell as defined in (g)(f), or A drug that specifically binds to MHC class I molecules expressed on the surface of cells as defined in (h)(f), including a soluble TCR, an antibody or its antigen-binding fragment, or a CAR, or a nucleic acid encoding the soluble TCR, antibody, its antigen-binding fragment, or CAR. 77. The drug for use described in item 76, wherein the cancer is melanoma. 78. A drug for use as described in item 76 or 77, further comprising using at least one additional antitumor agent or therapy on the subject. 79. The agents for use described in item 78, wherein the at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiotherapy, or surgery.

[0009] Other purposes, advantages, and features of this disclosure will become more apparent from reading the following non-restrictive description of specific embodiments, which are given only as examples with reference to the accompanying drawings. [Brief explanation of the drawing]

[0010] [Figure 1A] Figures 1A–1E demonstrate that aeTSA is immunogenic and may contribute to the response to ICB in melanoma. Box plots showing the number of TA-HLA pairs per pre-treatment sample (gray dots) from Riaz et al. 8, according to the response group from the original study. P values ​​from unpaired two-sided t-tests. [Figure 1B] This study demonstrates that aeTSA is immunogenic and may contribute to the response to ICB in melanoma. Box plots showing the number of TA-HLA pairs in pre-treatment and in-treatment samples from Riaz et al. 8, according to the response group from the original study. The gray line connects the pre-treatment and in-treatment samples for each patient. P-values ​​from paired two-sided t-tests. [Figure 1C] This study demonstrates that aeTSA is immunogenic and may contribute to the response to ICB in melanoma. Pearson correlations between the number of proliferated T cell clones and the number of TA-HLA pairs lost during therapy per patient (colored dots) are shown, according to the response group from the original study (Riaz et al. 8). Patients with stable disease (SD) were excluded due to the small number of samples (n=2) with both RNA-seq and TCR-seq data. [Figure 1D] This demonstrates that aeTSA is immunogenic and may contribute to the response to ICB in melanoma. The CD8 T cell chronotypes proliferated in treatment samples from responder Pt44 in the Riaz et al. cohort 8 are shown as percentages of all chronotypes. Here, the IGF2BP1-derived aeTSA IMKKIRESY-specific TCRB chronotype is highlighted. The gray lines connect the respective proliferated TCRB chronotypes in pre-treatment and treatment samples (left) and the RNA expression of homologous aeTSA in pre-treatment and treatment samples from Pt44 (right). [Figure 2A] This shows predictions of non-mutant TA presentation in melanoma and NSCLC samples from patients who underwent ICB. Box plots show the number of TA-HLA pairs per pre-treatment sample (gray dots) (i.e., the total number of HLA alleles per sample capable of presenting each expressed TA) from various published studies in melanoma11,12,19–21, according to response group from the original studies. P-values ​​from unpaired two-sided t-tests are also shown. No adjustments were made for multiple testing. [Figure 2B] This shows the prediction of non-mutant TA presentation in melanoma and NSCLC samples from patients who underwent ICB. Box plots showing the number of TA-HLA pairs in pre-treatment and in-treatment samples from Gide et al. 11 (Figure 2B) and Du et al. 12 (Figure 2C), according to the response group from the original study. The gray line connects the pre-treatment and in-treatment samples for each patient. P-values ​​from paired two-tailed t-tests are shown. No adjustment for multiple testing was performed. [Figure 2C] This shows the prediction of non-mutant TA presentation in melanoma and NSCLC samples from patients who underwent ICB. Box plots showing the number of TA-HLA pairs in pre-treatment and in-treatment samples from Gide et al. 11 (Figure 2B) and Du et al. 12 (Figure 2C), according to the response group from the original study. The gray line connects the pre-treatment and in-treatment samples for each patient. P-values ​​from paired two-tailed t-tests are shown. No adjustment for multiple testing was performed. [Figure 2D] This shows predictions of non-mutant TA presentation in melanoma and NSCLC samples from patients who underwent ICB. The box plot shows the difference in purity scores from ESTIMATE between in-therapy and pre-therapy samples. Here, negative values ​​indicate a decrease in tumor purity during therapy in samples from Riaz et al. 8 (top). The lower heatmap shows the Pearson correlation between the change in purity (from the upper panel) and the change in the number of TA-HLA pairs in in-ICB samples compared to pre-ICB samples from corresponding patients. The box plots show the median and interquartile range (IQR), with whiskers extending from the box hinge to a maximum value not exceeding 1.5 × IQR. [Figure 3A] The results of a FEST assay showing proliferation of specific T cell chronotypes after in vitro stimulation by aeTSA, selected based on the complete loss of RNA expression in at least one responder during therapy, as described in Riaz et al.8. [Figure 3B] This document demonstrates that the immunogenicity predictor PRIME2.0 identifies melanoma aeTSA as an immunogenic target. For the peptide sequences of aeTSA (n=200 binding to 27 HLA alleles) and mTSA (n=5 binding to 5 HLA alleles), as well as immunogenic (n=590 binding to 53 HLA alleles) and non-immunogenic (n=5887 binding to 62 HLA alleles), used to train PRIME2.0, the PRIME score for each peptide-HLA allele pair was calculated by PRIME2.0. For each unique peptide sequence, the score of the highest-scoring HLA allele was retained and used for plotting. The groups were compared using the non-parametric Mann-Whitney U test, where ns represents non-significant and ***P-value < 1E-10. [Figure 4A] This shows TA sharing and expression regulation across cancer samples. The stacked bar graph shows the proportion of TA types (and absolute number of TAs) shared across different numbers of melanoma samples analyzed. [Figure 4B]This shows TA sharing and expression regulation across cancer samples. The box plot shows the percentage of TCGA samples expressing each TA (gray dot) at at least twice the 95th percentile value of each TA in GTEx samples excluding testicular melanoma TAs. The box plot shows the median and interquartile range (IQR), with whiskers extending from the box hinge to a maximum value not exceeding 1.5 × IQR. [Figure 4C] This demonstrates TA sharing and expression regulation across cancer samples. Spearman correlations (Figure 4C) between RPHM expression of each melanoma TA across analyzed SKCM samples from TCGA and corresponding omics values ​​[FPKM expression, copy number variation, methylated β-value, and tumor mutational burden (TMB)] are shown, as well as the proportion of TAs with significant correlations (p-adj < 0.05, among TAs with available omics data, shown as a heatmap of cells marked with * in Figure 4C (non-empty cells in Figure 4C) (Figure 4D). [Figure 4D] This demonstrates TA sharing and expression regulation across cancer samples. Spearman correlations (Figure 4C) between RPHM expression of each melanoma TA across analyzed SKCM samples from TCGA and corresponding omics values ​​[FPKM expression, copy number variation, methylated β-value, and tumor mutational burden (TMB)] are shown, as well as the proportion of TAs with significant correlations (p-adj < 0.05, among TAs with available omics data, shown as a heatmap of cells marked with * in Figure 4C (non-empty cells in Figure 4C) (Figure 4D). [Figure 5] Box plots showing stem cell scores obtained using ssGSEA in TCGA samples studied herein across the LUAD, LUSC, and SKCM cohorts are shown. P-values ​​from the two-sided Wilcoxon test are also shown. [Figure 6A] Annotation of scRNA-seq data from previous studies of melanoma is shown. A balloon plot showing the mean expression and percentage of cells expressing the indicated gene is used for cluster annotation in each cluster identified across cutaneous melanoma samples from Zhang et al.14. The genes used for cluster annotation are derived from the original paper. [Figure 6B]Annotation of scRNA-seq data from previous studies of melanoma is shown. UMAP of their cell type annotations (bottom) according to identified clusters (top) and (bottom) genes across cutaneous melanoma samples from Zhang et al.14. [Figure 7A] This shows the expression of non-mutant TA in scRNA-seq data from melanoma. The box plot from Figure 8A shows the read count of cancer-specific melanoma TA across cell types from cutaneous melanoma samples from Zhang et al.14. Each gray dot represents one TA per cell. [Figure 7B] This shows the expression of non-mutant TA in scRNA-seq data from melanoma. The box plot shows the read count of melanoma TA expressed in non-cancer cell types from cutaneous melanoma samples from Zhang et al.14. Each gray dot represents one TA per cell. [Figure 8A] This shows the expression of TA in scRNA-seq data from melanoma. The bar graph shows the percentage (and absolute number) of melanoma TA expressed only in cancer cells (read counts greater than 1) in cutaneous melanoma scRNA-seq data from Zhang et al.14. [Figure 8B] This shows TA expression in scRNA-seq data from melanoma. The percentage of cell doublets in the TA-negative cell fraction relative to TA-expressing cells (cells expressing TA > 1 read count) for each annotated cell type from cutaneous melanoma, from Zhang et al.14. Each gray dot represents TA expressed in at least one cell of each cell type. [Figure 8C]This shows TA expression in scRNA-seq data from melanoma. The box plots show the normalized expression of MLANA (upper panel) and PMEL (lower panel) in cell types derived from cutaneous melanoma samples, comparing cells expressing at least one TA (TApos) to cells negative for all TAs (TAneg) for each TA type identified in melanoma. All box plots show the median and interquartile range (IQR), with whiskers extending from the box hinge to a maximum value not exceeding 1.5 × IQR. P-values ​​from the two-sided Wilcoxon nonparametric test are shown. No adjustments were made for multiple testing. [Modes for carrying out the invention]

[0011] In the context of describing the technology (particularly in the context of the following claims), the terms “a,” “an,” and “the,” as well as similar referents, are to be interpreted as encompassing both singular and plural forms unless otherwise indicated herein or unless it is clearly inconsistent with the context.

[0012] The terms “comprising,” “having,” “including,” and “containing” are to be interpreted as open-ended terms (i.e., “including, but not limited to”) unless otherwise noted.

[0013] All methods described herein may be carried out in any preferred order, unless otherwise indicated herein or unless it is clearly inconsistent with the context.

[0014] Any and all embodiments provided herein, or the use of exemplary language ("e.g.", "etc."), are intended solely to better illustrate the claimed embodiments of the technology and, unless otherwise claimed, do not constitute a limitation of scope.

[0015] Nothing expressed herein should be construed as indicating that any non-claimed element is essential to the implementation of the claimed embodiment of the technology.

[0016] In this specification, the term “about” has its usual meaning. The term “about” is used to indicate that a value includes inherent variability due to errors in the device or method used to determine the value, or that it includes values ​​close to the enumerated values, for example, values ​​within 10% of the enumerated values ​​(or range of values).

[0017] The enumeration of value ranges in this specification is intended, unless otherwise indicated herein, simply as a concise way of referring individually to each distinct value within that range, and each distinct value is incorporated herein as if it were individually listed herein. All subsets of values ​​within a range are also incorporated herein as if they were individually listed herein.

[0018] If any feature or aspect of the present disclosure is described in terms of the Markush group or list of substitutes, a person skilled in the art will recognize that the present disclosure is also described in terms of any individual component or subgroup of components of the Markush group or list of substitutes.

[0019] Unless otherwise specifically defined, all technical and scientific terms used herein should be interpreted as having the same meaning as commonly understood by those skilled in the art (for example, in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0020] Unless otherwise indicated, the recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FMAusubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all revisions to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JEColigan et al. (editors), Current This is described and explained throughout the literature in sources such as *Protocols in Immunology*, John Wiley & Sons (including all revisions to date).

[0021] In the studies described herein, the inventors identified 505 tumor antigens (TAs) discovered using comprehensive proteogenetic analysis of cutaneous melanoma. Of these 505 TAs, 5 (1%) originated from mutant sequences and 500 (99%) originated from non-mutant genomic sequences. Mutant TAs were rare and patient-specific, while non-mutant TAs were shared among tumor samples. Non-mutant TAs belong to three classes: i) 200 aberrantly-expressed tumor-specific antigens (aeTSA) not present in normal tissue; ii) 129 tumor-associated antigens (TAAs) overexpressed in tumors compared to normal tissue; and iii) 171 lineage-specific antigens (LSA) specifically expressed in melanoma samples, normal skin, and melanocytes. The majority of TAAs (88%) originate from exons of annotated protein-coding genes, while approximately half of aeTSAs (53%) and approximately half of LSAs (45%) originate from genomic regions considered to be non-coding genomic regions, such as ncRNAs, introns, and intergenetic regions. The novel tumor antigen candidates identified herein, including melanocyte lineage-specific antigens (LSAs), tumor-associated antigens (TAAs), and tumor-specific antigens (TSAs), may be useful in immunotherapies and vaccines for cancers expressing tumor antigen candidates, such as melanoma.

[0022] This disclosure relates to a tumor antigen peptide (TAP) (or tumor-specific peptide), such as a melanoma TAP, comprising or consisting of one of the amino acid sequences defined in any one of Sequence IDs 1 to 505. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0023] Generally, peptides such as tumor antigen peptides (TAPs) presented in relation to HLA class I vary in length by about 7 or 8 to about 15, or preferably 8 to 14, amino acid residues. In some embodiments of the methods of this disclosure, longer peptides, including the TAP sequence as defined herein, are artificially loaded onto cells such as antigen-presenting cells (APCs) processed by cells, and the TAP is presented on the surface of the APC by an MHC class I molecule. In this method, peptides / polypeptides longer than 15 amino acid residues can be loaded onto the APC and processed by a protease in the cytoplasm of the APC, providing the corresponding TAP as defined herein for presentation. In some embodiments, the precursor peptide / polypeptide used to generate the TAP as defined herein is, for example, 1000, 500, 400, 300, 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20, or 15 or fewer amino acids. Therefore, all methods and processes using TAPs described herein involve the use of longer peptides or polypeptides (including native proteins), i.e., tumor antigen precursor peptides / polypeptides, to induce the presentation of "final" 8-14 TAPs after processing by cells (APCs). In some embodiments, the TAPs described herein are about 8-14, 8-13, or 8-12 amino acid lengths (e.g., 8, 9, 10, 11, 12, or 13 amino acid lengths) and small enough to fit directly into HLA class I molecules. In one embodiment, the TAP contains 20 or fewer amino acids, preferably 15 or fewer, more preferably 14 or fewer. In one embodiment, the TAP contains at least 7 amino acids, preferably at least 8 or fewer, more preferably at least 9 amino acids.

[0024] As used herein, the term “amino acid” includes both L-isomers and D-isomers of naturally occurring amino acids used in peptide chemistry for preparing synthetic analogs of TAP, as well as other amino acids (e.g., naturally occurring amino acids, naturally occurring amino acids, amino acids not encoded by nucleic acid sequences, etc.). Examples of naturally occurring amino acids include glycine, alanine, valine, leucine, isoleucine, serine, and threonine. Other amino acids include, for example, non-gene-coding amino acids, amino acid analogs, and conserved substitutions of L-amino acids. Naturally occurring non-genetically coded amino acids and amino acid analogs include, for example, beta-alanine, 3-aminopropionic acid, 2,3-diaminopropionic acid, alpha-aminoisobutyric acid (Aib), 4-aminobutyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine (Nle), norvaline, 2-naphthylalanine, pyridylalanine, 3-benzothienylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3- Examples include fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, beta-2-thienylalanine, methionine sulfoxide, L-homoarginine (Hoarg), N-acetyllysine, 2-aminobutyric acid, 2-aminobutyric acid, 2,4-diaminobutyric acid (D- or L-), p-aminophenylalanine, N-methylvaline, homocysteine, homoserine (HoSer), cysteic acid, epsilon-aminohexanoic acid, delta-aminovaleric acid, benzyloxytyrosine, β-phenylalanine, or 2,3-diaminobutyric acid (D- or L-). These amino acids are well known in the fields of biochemistry / peptide chemistry. Therefore, one or more of the amino acids in the TAPs (SEQ ID NOs. 1-505) described herein may be replaced by non-gene-coding amino acids and / or amino acid analogs.TAP may also be modified to improve the proteolytic stability of the peptide by incorporating, for example, methyl amino acids, β-amino acids, or peptoids. In one embodiment, TAP contains only naturally occurring amino acids.

[0025] In several embodiments, the TAPs described herein include peptides having modified sequences that include functionally equivalent amino acid residue substitutions compared to the sequences described herein. For example, one or more amino acid residues in a sequence may be substituted with another amino acid of similar polarity (having similar physicochemical properties) that acts as a functional equivalent, resulting in a silent modification. The amino acid substitutions in a sequence may be selected from other members of the class to which the amino acid belongs. For example, positively charged (basic) amino acids include arginine, lysine, and histidine (as well as homoarginine and ornithine). Nonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophan, and methionine. Uncharged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Negatively charged (acidic) amino acids include glutamic acid and aspartic acid. The amino acid glycine may belong to either the nonpolar amino acid family or the uncharged (neutral) polar amino acid family. Substitutions made within an amino acid family are generally understood to be conservative substitutions. The TAPs described herein may contain all L-amino acids, all D-amino acids, or mixtures of L-amino acids and D-amino acids. In one embodiment, the TAPs described herein contain all L-amino acids.

[0026] In one embodiment, in a TAP sequence containing or consisting of one of the sequences of SEQ ID NOs: 1 to 505, amino acid residues that do not substantially contribute to interaction with the T cell receptor may be modified by replacing them with other amino acids whose incorporation does not substantially affect T cell reactivity and does not eliminate binding to the relevant MHC.

[0027] TAPs may also be modified by replacing one or more amide bonds of the peptide, which can improve chemical stability and / or enhance biological / pharmacological properties (e.g., half-life, absorption, potency, efficiency, etc.). Typical peptide bond substitutions include esters, polyamines and their derivatives, as well as substituted alkanes and alkenes such as aminomethyl and ketomethylenes. For example, the above-mentioned TAPs may have one or more amide bonds replaced by linkages such as -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH-(cis or trans), -CH2SO-, -CH(OH)CH2-, or -COCH2-.

[0028] TAP may also be capped or modified at its N-terminus and / or C-terminus to prevent degradation and improve stability, affinity, and / or uptake. Thus, in another embodiment, the present disclosure relates to formula Z 1 -XZ 2 The formula provides a modified TAP, where X is a TAP containing or consisting of one of the amino acid sequences of sequence numbers 1 to 505.

[0029] In one embodiment, the amino-terminal residue of TAP (i.e., the free amino group at the N-terminus) is, for example, a partial / chemical group (Z 1 ) is modified by covalent bonds (for example, for protection against decomposition). Z 1 C1-C2 may be a linear or branched alkyl group of 1-8 carbon atoms, or an acyl group (R-CO-), where R is a hydrophobic moiety (e.g., acetyl, propionyl, butanyl, isopropionyl, or isobutanyl) or an alloyl group (Ar-CO-), where Ar is an aryl group. In one embodiment, the acyl group is C1-C2 16 Or C3~C 16It is an acyl group (linear or branched, saturated or unsaturated), and in a further embodiment, it is a saturated C1-C6 acyl group (linear or branched) or an unsaturated C3-C6 acyl group (linear or branched), for example, an acetyl group (CH3-CO-, Ac). In one embodiment, Z 1 is absent. The carboxy-terminal residue of TAP (i.e., the free carboxy group at the C-terminus of TAP) may be modified, for example, by amidation (replacement of the OH group by an NH2 group) (e.g., for protection against degradation), and thus, in such a case, Z 2 is an NH2 group. In one embodiment, Z 2 may be a hydroxamate group, a nitrile group, an amide (primary, secondary, or tertiary) group, an aliphatic amine of 1 to 10 carbons such as methylamine, iso-butylamine, iso-valeryl amine, or cyclohexylamine, an aromatic or arylalkylamine such as aniline, naphthylamine, benzylamine, cinnamylamine, or phenylethylamine, an alcohol, or CH2OH. In one embodiment, Z 2 is absent. In one embodiment, TAP comprises one of the amino acid sequences of SEQ ID NOs: 1 to 505. In one embodiment, TAP consists of one of the amino acid sequences of SEQ ID NOs: 1 to 505, i.e., Z 1 and Z 2 are absent.

[0030] In another aspect, the present disclosure provides a TAP that binds to the HLA-A*01:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 11, 14, 20, 27, 29, 37, 86, 87, 121, 150, 165, 173, 279, 288, 299, 3o8, 312, 316, 319, 331, 340, 352, 362, 363, 384, $95, 398, 411, 419, 429, 458, 474, 476, 487, 492, 500, or 501.

[0031] In another embodiment, the present disclosure binds to the HLA-A*02:01 molecule, and includes SEQ ID NOs: 1, 2, 4, 19, 22, 25, 40, 50, 52, 54, 60, 65, 67, 76, 81, 89, 91, 103, 107, 120, 124, 128, 135, 157, 175, 179, 186, 188, 201, 208, 218, 219, 228, 232, 233, 240, 2 Provide a TAP containing or consisting of the following sequences: 50, 257, 261, 263, 265, 266, 282, 286, 298, 311, 315, 317, 325, 333, 337, 349, 367, 373, 387, 388, 393, 399, 404, 412, 414, 415, 422, 434, 437, 455, 480, 489, or 494.

[0032] In another embodiment, the Disclosure provides a TAP that binds to an HLA-A*02:09 molecule and comprises or consists of the sequences of SEQ ID NOs: 22, 40, 50, 62, 83, 89, 232, 265, 282, 311, 315, 317, 337, 349, 367, 399, 407, 412, or 494.

[0033] In another embodiment, the Disclosure provides a TAP that binds to an HLA-A*03:01 molecule and comprises or consists of the sequences of SEQ ID NOs. 209, 247, 272, 347, or 505.

[0034] In another embodiment, the Disclosure provides a TAP that binds to an HLA-A*23:01 molecule and comprises or consists of the sequences of SEQ ID NOs: 48, 84, 129, 151, 152, 171, 174, 182, 223, 237, 245, 290, 358, 397, 426, 446, 481, 482, or 486.

[0035] In another embodiment, the present disclosure provides a TAP that binds to an HLA-A*24:02 molecule and comprises or consists of the sequence of SEQ ID NOs: 31, 244, or 481.

[0036] In another embodiment, the present disclosure provides a TAP that binds to an HLA-A*25:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 11, 31, 249, 289, 403, 425, 464, or 490.

[0037] In another embodiment, the Disclosure provides a TAP that binds to an HLA-A*26:01 molecule and comprises or consists of the sequences of SEQ ID NOs: 11, 20, 27, 39, 41, 42, 45, 114, 116, 122, 168, 181, 268, 284, 329, 364, 398, 417, 430, 440, 447, 452, 457, 460, 462, 475, 490, or 497.

[0038] In another embodiment, the Disclosure provides a TAP that binds to an HLA-A*30:01 molecule and comprises or consists of the sequence of SEQ ID NOs. 26, 304, or 376.

[0039] In another embodiment, the Disclosure provides a TAP that binds to an HLA-A*30:02 molecule and comprises or consists of the sequences of SEQ ID NOs: 117, 185, 190, 193, 197, 256, 294, 323, 471, or 485.

[0040] In another embodiment, the Disclosure provides a TAP that binds to an HLA-A*32:01 molecule and comprises or consists of the sequences of SEQ ID NOs: 17, 44, 46, 61, 95, 106, 126, 143, 170, 194, 208, 221, 264, 296, 324, 326, 401, 414, 420, 425, or 469.

[0041] In another embodiment, the Disclosure provides a TAP that binds to an HLA-A*33:01 molecule and comprises or consists of the sequence of SEQ ID NO: 248.

[0042] In another embodiment, the present disclosure binds to the HLA-A*68:01 molecule and includes SEQ ID NOs: 6-8, 12, 15, 23, 28, 41, 42, 47, 51, 53, 56, 59, 64, 66, 68, 70, 73-75, 77, 79, 80, 82, 85, 88, 92, 93, 96-99, 101, 102, 108-112, 1 18, 119, 123, 130, 131, 134, 137, 138, 141, 142, 144~147, 149, 153, 154, 156, 158, 160, 161, 166, 169, 172, 176, 184, 198, 202, 203, 212, 214, 216, 217, 220, 224, 226, 2 29, 234, 236, 239, 248, 254, 269, 270, 275, 280, 283, 287, 293, 300, 301, 303, 306, 310, 314, 320, 328, 332, 348, 354, 355, 359, 360, 366, 369, 370, 372, 381, 382, ​​386, 3 Provide a TAP containing or consisting of the sequence 89, 390, 392, 394, 402, 405, 406, 409, 413, 418, 424, 428, 436, 441~445, 449, 456, 459, 465~468, 473, 479, 483, 488, 493, 499, or 502.

[0043] In another embodiment, the Disclosure provides a TAP that binds to an HLA-B*07:02 molecule and comprises or consists of the sequences of SEQ ID NOs: 35, 36, 43, 104, 125, 127, 139, 140, 187, 191, 196, 206, 231, 243, 246, 251, 252, 260, 281, 291, 292, 321, 330, 361, 408, 432, 435, 439, 451, 461, 491, or 503.

[0044] In another embodiment, the present disclosure provides a TAP that binds to an HLA-B*08:01 molecule and comprises or consists of the sequences of SEQ ID NOs: 21, 162, 189, 286, 307, 309, 317, 339, 343, 349, or 414.

[0045] In another embodiment, the Disclosure provides a TAP that binds to an HLA-B*13:02 molecule and comprises or consists of the sequence of SEQ ID NOs. 207, 311, 393, 400, or 455.

[0046] In another embodiment, the present disclosure provides a TAP that binds to an HLA-B*14:01 molecule and comprises or consists of the sequence of SEQ ID NO: 396.

[0047] In another embodiment, the Disclosure provides a TAP that binds to an HLA-B*15:01 molecule and comprises or consists of the sequences of SEQ ID NOs: 11, 26, 30, 63, 113, 132, 148, 192, 195, 200, 207, 210, 222, 235, 238, 258, 262, 267, 276, 295, 297, 305, 309, 318, 326, 336, 342, 374, 377, 383, 401, 421, 427, 433, 438, 450, 463, 470, or 477.

[0048] In another embodiment, the Disclosure provides a TAP that binds to an HLA-B*18:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 3, 10, 33, 225, or 396.

[0049] In another embodiment, the present disclosure provides a TAP that binds to an HLA-B*35:03 molecule and comprises or consists of the sequences of SEQ ID NOs: 5, 16, 100, 155, 164, 211, 230, 253, 259, 277, 327, or 335.

[0050] In another embodiment, the Disclosure provides a TAP that binds to an HLA-B*37:01 molecule and comprises or consists of the sequence of SEQ ID NO: 302.

[0051] In another embodiment, the Disclosure provides a TAP that binds to an HLA-B*38:01 molecule and comprises or consists of the sequences of SEQ ID NOs: 26, 180, 338, 341, 378, 380, 416, 448, 454, 496, or 498.

[0052] In another embodiment, the present disclosure provides a TAP that binds to an HLA-B*40:01 molecule and comprises or consists of the sequence of SEQ ID NO: 215 or 322.

[0053] In another embodiment, the present disclosure provides a TAP that binds to an HLA-B*40:02 molecule and comprises or consists of the sequence of SEQ ID NOs. 213, 215, 339, or 351.

[0054] In another embodiment, the Disclosure provides a TAP that binds to an HLA-B*44:02 molecule and comprises or consists of the sequence of SEQ ID NOs: 346, 353, 356, or 453.

[0055] In another embodiment, the Disclosure provides a TAP that binds to an HLA-B*49:01 molecule and comprises or consists of the sequences of SEQ ID NOs: 9, 57, 78, 115, 136, 167, 255, 273, 274, 278, 313, 339, 480, 484, or 495.

[0056] In another embodiment, the present disclosure provides a TAP that binds to an HLA-B*51:01 molecule and comprises or consists of the sequence of SEQ ID NO: 227 or 259.

[0057] In another embodiment, the Disclosure provides a TAP that binds to an HLA-C*03:03 molecule and comprises or consists of the sequence of SEQ ID NOs: 24, 69, 204, or 388.

[0058] In another embodiment, the Disclosure provides a TAP that binds to an HLA-C*03:04 molecule and comprises or consists of the sequences of SEQ ID NOs: 24, 49, 69, 159, 241, 242, or 334.

[0059] In another embodiment, the present disclosure provides a TAP that binds to an HLA-C*04:01 molecule and comprises or consists of the sequence of SEQ ID NOs. 90, 257, or 423.

[0060] In another embodiment, the Disclosure provides a TAP that binds to an HLA-C*06:02 molecule and comprises or consists of the sequence of SEQ ID NOs: 13, 34, or 72.

[0061] In another embodiment, the Disclosure provides a TAP that binds to an HLA-C*07:01 molecule and comprises or consists of the sequences of SEQ ID NOs: 13, 75, 133, 163, 183, 199, 205, 285, 334, 391, or 472.

[0062] In another embodiment, the Disclosure provides a TAP that binds to an HLA-C*07:02 molecule and comprises or consists of the sequence of SEQ ID NOs: 13, 32, 36, 105, 177, 339, 371, or 478.

[0063] In another embodiment, the Disclosure provides a TAP that binds to an HLA-C*08:02 molecule and comprises or consists of the sequence of SEQ ID NOs: 18, 54, or 379.

[0064] In another embodiment, the present disclosure provides a TAP that binds to an HLA-C*12:03 molecule and comprises or consists of the sequences of SEQ ID NOs: 24, 49, 58, 71, 94, 162, 178, 242, 289, 344, 345, 357, 367, 368, 375, 423, or 431.

[0065] In another embodiment, the Disclosure provides a TAP that binds to an HLA-C*14:02 molecule and comprises or consists of the sequences of SEQ ID NOs: 105, 271, 350, 365, 385, 410, 423, or 504.

[0066] The TAPs of this disclosure may be produced by expression in host cells containing nucleic acids encoding TAP (recombinant expression), or by chemical synthesis (e.g., solid-phase peptide synthesis). The peptides can be readily synthesized by manual and / or automated solid-phase procedures well known in the art. Preferred synthesis can be carried out, for example, by utilizing the "T-boc" or "Fmoc" procedures. Techniques and procedures for solid-phase synthesis are described, for example, in Solid Phase Peptide Synthesis: A Practical Approach, E. Atherton and RC Sheppard, IRL, Oxford University Press, 1989. Alternatively, TAP can be used as described in, for example, Liu et al., Tetrahedron Lett.37:933-936,1996, Baca et al., J.Am.Chem.Soc.117:1881-1887,1995, Tam et al., Int. 256:221-225,1992, Liu and Tam, J.Am.Chem.Soc.116:4149-4153,1994, Liu and Tam,Proc.Natl.Acad.Sci.USA 91:6584-6588,1994, and Yamashiro and Li,Int.J.Peptide Protein TAP may be prepared by segment condensation, as described in Res.31:322-334, 1988). Other methods useful for the synthesis of TAP are described in Nakagawa et al., J.Am.Chem.Soc.107:7087-7092, 1985. In one embodiment, TAP is chemically synthesized (synthetic peptide). Another embodiment of the present disclosure relates to a peptide that does not exist in nature, which consists of or is essentially an amino acid sequence as defined herein and is produced synthetically (e.g., synthesized) as a pharmaceutically acceptable salt. The salts of TAP according to the present disclosure are substantially different from the peptides in vivo, since the peptides produced in vivo do not have salts.Non-natural salt forms of peptides may, specifically, modulate the solubility of peptides in the context of pharmaceutical compositions, such as peptide vaccines disclosed herein. Preferably, the salt is a pharmaceutically acceptable salt of the peptide.

[0067] In one embodiment, the TAP described herein is substantially pure. A compound is "substantially pure" when it is separated from its naturally occurring components. Typically, a compound is substantially pure when it contains at least 60%, more commonly 75%, 80%, or 85%, preferably more than 90%, and more preferably more than 95%, per weight of the total material in the sample. Thus, for example, polypeptides that are chemically synthesized or produced by recombinant technology will generally contain substantially no components associated with their natural origin, such as the polymer components of their source. A nucleic acid molecule is substantially pure when it is not immediately contiguous (i.e., covalently bonded) to a coding sequence that is normally contiguous in the naturally occurring genome of the organism from which the nucleic acid originates. Substantially pure compounds can be obtained, for example, by extraction from a natural source, by expression of recombinant nucleic acid molecules encoding a peptide compound, or by chemical synthesis. Purity can be measured using any suitable method such as column chromatography, gel electrophoresis, or HPLC. In one embodiment, the TAP is in solution. In another embodiment, the TAP is in solid form, for example, lyophilized.

[0068] In one embodiment, TAP is encoded by a sequence located in a non-protein-coding region of the genome. In another embodiment, TAP is encoded by a sequence located in an intergeneric region. In yet another embodiment, TAP is encoded by non-coding RNA (ncRNA). In yet another embodiment, TAP is encoded by a sequence located in an intron. In yet another embodiment, TAP is encoded by a sequence located in an untranslated region (UTR), for example, the 5'-UTR. In yet another embodiment, TAP is encoded by a sequence located in a non-coding region.

[0069] In another embodiment, the disclosure further provides synthetic long peptides (SLPs) comprising at least one of the TAPs described herein. In one embodiment, the SLP comprises at least two TAPs, at least one of which is a TAP described herein. In one embodiment, the SLP comprises at least two, three, four, or five TAPs described herein. In one embodiment, the SLP comprises at least ten, fifteen, twenty, twenty-five, thirty, thirty-five, or forty TAPs described herein. In one embodiment, the SLP comprises at least one of the TAPs described herein, linked to one or more amino acid sequences or domains that confer desired properties to the SLP, such as sequences containing motifs that can be cleaved by cellular proteases such as cathepsins, thereby stabilizing the SLP and / or improving processing and presentation by MHC molecules. In another embodiment, the SLP comprises at least one of the TAPs described herein and a TAP that binds to an MHC class II molecule. TAPs may be directly linked to each other, or they may be indirectly linked via linkers such as short amino acid linkers. In several embodiments, the linker comprises about 4 to about 20 amino acids, or about 4 to about 15 amino acids, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In one embodiment, the linker comprises a glycine residue, a serine residue, a proline residue, a threonine residue, or a mixture thereof. The linker may also contain a sequence that facilitates the processing of the SLP to release TAP, for example, a cathepsin-sensitive linker (e.g., a 4-6 amino acid linker containing sequences LVGS (SEQ ID NO: 1138), ASLG (SEQ ID NO: 1139), PIVG (SEQ ID NO: 1140), LLSV (SEQ ID NO: 1141), VLSVG (SEQ ID NO: 1142), or LLSVGG (SEQ ID NO: 1143) (see Rabu et al., Oncoimmunology. 2019; 8(4):e1560919). In one embodiment, the SLP has a length of 200, 150, 100, 90, 80, 70, 60, or 50 or fewer amino acids.In further embodiments, the SLP has a length of 20 to 50, 45, or 40 amino acids, for example, 20 or 25 amino acids to 30, 35, or 40 amino acids.

[0070] The TAP or SLP described herein may further include one or more modifications that confer additional biological properties to the TAP or SLP, such as protease resistance, plasma protein binding, increased plasma half-life, and intracellular penetration. Such modifications include, for example, fatty acids (e.g., C6-C6). 18 This includes covalent bonding of molecules / parts such as ) to the TAP or SLP, binding of proteins such as albumin (see, for example, U.S. Patent No. 7,268,113), sugar / polysaccharide (glycosylation), biotinylation or PEGylation (see, for example, U.S. Patents No. 7,256,258 and 6,528,485). The above description of modifications of the TAP or SLP does not limit the scope of the approach or the possible modifications that can be manipulated. Accordingly, in another aspect, this disclosure provides a conjugate comprising the TAP or SLP described herein and one or more additional molecules or agents (hereinafter, secondary molecules or agents). The TAP or SLP may be conjugated to any kind of synthetic or natural secondary molecule or agent, such as peptides, proteins, sugars / polysaccharides, lipids, naturally occurring polymers or synthetic polymers / copolymers, in order to modify one or more properties of the TAP or SLP.

[0071] In another embodiment, the Disclosure further provides (isolated) nucleic acids encoding TAP or tumor antigen precursor peptides or SLPs as described herein. In one embodiment, the nucleic acid comprises about 21 to about 45 nucleotides, about 24 to about 45 nucleotides, for example, 24, 27, 30, 33, 36, 39, 42, or 45 nucleotides. As used herein, “isolated” means a peptide or nucleic acid molecule isolated from other components present in the natural environment of the molecule or from macromolecules of naturally occurring sources (e.g., including other nucleic acids, proteins, lipids, sugars, etc.). As used herein, “synthesized” means a peptide or nuclear molecule that has not been isolated from its natural source, produced, for example, by recombinant technology or using chemical synthesis. In one embodiment, the nucleic acid (DNA, RNA) encoding TAP of the Disclosure comprises or consists of one of the sequences defined in Table 1 below or the corresponding RNA sequences. In one embodiment, the nucleic acid encoding TAP is an mRNA molecule. In other embodiments, the nucleic acid encoding TAP or SLP is self-amplified mRNA (saRNA), transcriptionally amplified mRNA (taRNA), or circular mRNA (circRNA) (see, for example, Liu et al., Nature Reviews Cancer, Volume 23, August 2023, pages 526–543). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11]

[0072] Of course, because the genetic code is degenerate, the TAPs described herein may be encoded by variants of the sequences mentioned above.

[0073] The nucleic acids of this disclosure may be used for recombinant expression of the TAP or SLP of this disclosure and may be contained in a vector or plasmid, such as a cloning vector or expression vector, which may be transfected into a host cell. In one embodiment, this disclosure provides a cloning vector, expression vector, or viral vector or plasmid containing a nucleic acid sequence encoding the TAP of this disclosure. Alternatively, the nucleic acid encoding the TAP of this disclosure may be incorporated into the genome of a host cell. In any case, the host cell expresses the TAP or protein encoded by the nucleic acid. As used herein, the term “host cell” refers not only to a specific target cell but also to the offspring or potential offspring of such a cell. The host cell may be any prokaryotic cell (e.g., E. coli) or eukaryotic cell (e.g., insect cell, yeast cell, plant cell, or mammalian cell) capable of expressing the TAP described herein. The vector or plasmid contains elements necessary for the transcription and translation of the inserted coding sequence and may contain other components such as resistance genes, cloning sites, etc. Expression vectors comprising a sequence encoding a peptide or polypeptide and appropriate transcriptional and translational regulatory / modulatory elements operably ligated thereto may be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, and Ausubel, FM et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY. "Operatably ligated" refers to the parallel arrangement of components, particularly nucleotide sequences, that enable them to perform their normal function. Thus, a coding sequence operably ligated to a regulatory sequence refers to a configuration of nucleotide sequences in which the coding sequence can be expressed under the regulatory control of the regulatory sequence, i.e., under transcriptional and / or translational control.As used herein, “regulatory / controllable region” or “regulatory / controllable sequence” refers to a non-coding nucleotide sequence involved in the regulation of coding nucleic acid expression. Therefore, the term regulatory region includes promoter sequences, regulatory protein binding sites, upstream activator sequences, etc. A vector (e.g., an expression vector) may have necessary 5' upstream and 3' downstream regulatory elements for efficient gene transcription and translation in its respective host cell, such as promoter sequences (e.g., CMV, PGK, and EF-1α promoters), ribosome recognition and binding TATA boxes, and 3'UTR AAUAAA transcription termination sequences. Other suitable promoters include constitutive promoters such as the Simiambimus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV) promoter, HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV very early promoter, and Rous sarcoma vims promoter. Human gene promoters may also be used, including, but not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. In certain embodiments, the inducible promoter is also intended to be part of a vector expressing TAP. This provides a molecular switch that can turn on or off the expression of the polynucleotide sequence of interest. Examples of inducible promoters include, but are not limited to, metallothione promoters, glucocorticoid promoters, progesterone promoters, or tetracycline promoters. Examples of vectors include plasmids, autonomous replication sequences, and transposition elements. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or Pl-derived artificial chromosomes (PACs)), bacteriophages (e.g., lambda phages or M13 phages), and animal viruses.Examples of animal virus categories useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors include the Lenti-X® Bicistronic Expression System (Neo) vector (Contech) and the pClneo vector (Promega) for expression in mammalian cells; and pLenti4 / V5-DEST®, pLenti6 / V5-DEST®, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequences of TAP disclosed herein can be ligated into such expression vectors for TAP expression in mammalian cells.

[0074] In certain embodiments, the nucleic acid encoding TAP of this disclosure is provided in a viral vector. The viral vector may be derived from an adenovirus, vaccinia virus, retrovirus, lentivirus, or formyvirus. As used herein, the term “viral vector” refers to a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be packaged into a viral vector particle. The viral vector may contain coding sequences for various proteins described herein instead of non-essential viral genes. In another embodiment, the nucleic acid encoding TAP of this disclosure is provided in a self-amplified or self-replicating RNA (srRNA) vector. The srRNA is derived from a positive-strand RNA virus from which structural proteins have been removed and replaced with the heterologous gene of interest. srRNA has been successfully derived from flaviviruses, nodamuraviruses, nidoviruses, and alphaviruses, which have therapeutic modes that provide trans-constituent structural proteins for the creation of single-cycle viral replicon particles (VRPs) (see, for example, Aliahmad et al. Next generation self-replicating RNA vectors for vaccines and immunotherapies. Cancer Gene Ther (2022). https: / / doi.org / 10.1038 / s41417-022-00435-8). Vectors and / or particles can be used for the purpose of transferring DNA, RNA, or other nucleic acids into cells, either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0075] In embodiments, the nucleic acids (DNA, RNA) encoding the TAPs of this disclosure are contained within vesicles or nanoparticles such as lipid vesicles (e.g., liposomes) or lipid nanoparticles (LNPs), or any other suitable vehicle. Thus, in another embodiment, this disclosure provides vesicles or nanoparticles such as lipid vesicles or nanoparticles containing nucleic acids such as mRNA encoding one or more of the TAPs described herein.

[0076] As used herein, the term liposome refers, in its usual sense, to a microscopic lipid vesicle composed of a phospholipid bilayer or any similar amphiphilic lipid (e.g., sphingolipid) that encapsulates an internal aqueous medium.

[0077] The term "lipid nanoparticles" refers to liposome-like structures that may include one or more lipid bilayer rings surrounding an internal aqueous medium similar to that of liposomes, or micelle-like structures encapsulating molecules (e.g., nucleic acids) within a non-aqueous core. Lipid nanoparticles typically contain cationic lipids, such as ionizable cationic lipids. Examples of cationic lipids that may be used in LNPs include DOTMA, DOSPA, DOTAP, ePC, DLin-MC3-DMA, C12-200, ALC-0315, cKK-E12, Lipid H(SM-102), OF-Deg-Lin, A2-Iso5-2DC18, and 306O i10 Examples include BAME-O16B, TT3, 9A1P9, FTT5, COATSOME® SS-E, COATSOME® SS-EC, COATSOME® SS-OC, and COATSOME® SS-OP (see, for example, Hou et al., Nature Reviews Materials, volume 6, pages 1078-1094 (2021), and Tenchov et al., ACS Nano, 15, 16982-17015 (2021)).

[0078] Liposomes and lipid nanoparticles typically contain lipids, lipid-like materials, and other lipid components such as polymers that can improve the properties of liposomes or nanoparticles, such as stability, delivery effectiveness, tolerance, and biodistribution. These include phospholipids such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol), as well as DOPE, sterols (such as cholesterol and cholesterol derivatives), and 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG). 2000 -DMG) and 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG 2000 It contains PEGylated lipids (PEG lipids) such as DSG.

[0079] In one embodiment, the lipid nanoparticles described herein comprise one or more cationic lipids, such as ionizable cationic lipids. Examples of ionizable cationic lipids include those listed in PCT Publications WO2017 / 061150 and WO2019 / 188867, which are commercially available under the trademark names COATSOME® SS-E, COATSOME® SS-EC, COATSOME® SS-OC, and COATSOME® SS-OP.

[0080] Nucleic acids encoding one or more of the TAPs (e.g., mRNA) may be modified, for example, to increase stability and / or reduce immunogenicity. For example, the 5' end may be capped (as described, for example, in US10519189 and US10494399) to stabilize the molecule and reduce immunogenicity. One or more nucleosides of mRNA may be modified or substituted with 1-methylpseudridine to either increase the stability of the molecule or reduce recognition of the molecule by the innate immune system. Forms of modified nucleosides are described in US9371511. Other types of modifications that may be performed on mRNA include anti-reverse cap analogs (ARCA), 5'-methylcytidine triphosphate (m5CTP), N6-methyladenosine-5'-triphosphate (m6ATP), 2-thiouridine triphosphate (s2UTP), pseudouridine triphosphate, N 1 Examples include the incorporation of methylpseudridine triphosphate or 5-methoxyuridine triphosphate (5moUTP). mRNA may also include additional modifications to the 5' and / or 3' untranslated region (UTR) and the polyadenylated (poly-A) tail (see, e.g., Kim et al., Molecular & Cellular Toxicology vol.18,1(2022):1-8). All of these and other modifications to nucleic acids encoding TAP (e.g., mRNA) are included in this disclosure.

[0081] In another aspect, the present disclosure provides an MHC class I molecule that includes (i.e., presents or is conjugated to) one or more TAPs comprising or consisting of the sequences of SEQ ID NOs: 1 to 505 as defined herein.

[0082] In one embodiment, the MHC class I molecule is the HLA-A*01:01 molecule. In one embodiment, the MHC class I molecule is the HLA-A*02:01 molecule. In one embodiment, the MHC class I molecule is the HLA-A*02:09 molecule. In one embodiment, the MHC class I molecule is the HLA-A*03:01 molecule. In one embodiment, the MHC class I molecule is the HLA-A*03:01 molecule. In one embodiment, the MHC class I molecule is the HLA-A*23:01 molecule. In one embodiment, the MHC class I molecule is the HLA-A*24:02 molecule. In one embodiment, the MHC class I molecule is the HLA-A*25:01 molecule. In one embodiment, the MHC class I molecule is the HLA-A*26:01 molecule. In one embodiment, the MHC class I molecule is the HLA-A*30:01 molecule. In one embodiment, the MHC class I molecule is an HLA-A*30:02 molecule. In one embodiment, the MHC class I molecule is an HLA-A*33:01 molecule. In one embodiment, the MHC class I molecule is an HLA-A*68:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*07:02 molecule. In one embodiment, the MHC class I molecule is an HLA-B*08:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*13:02 molecule. In one embodiment, the MHC class I molecule is an HLA-B*14:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*15:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*18:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*35:03 molecule. In one embodiment, the MHC class I molecule is the HLA-B*37:01 molecule. In one embodiment, the MHC class I molecule is the HLA-B*38:01 molecule. In one embodiment, the MHC class I molecule is the HLA-B*40:01 molecule. In one embodiment, the MHC class I molecule is the HLA-B*40:02 molecule. In one embodiment, the MHC class I molecule is the HLA-B*44:02 molecule. In one embodiment, the MHC class I molecule is the HLA-B*49:01 molecule. In one embodiment, the MHC class I molecule is the HLA-B*51:01 molecule. In one embodiment, the MHC class I molecule is the HLA-C*03:03 molecule.In one embodiment, the MHC class I molecule is HLA-C*03:04. In one embodiment, the MHC class I molecule is HLA-C*04:01. In one embodiment, the MHC class I molecule is HLA-C*06:02. In one embodiment, the MHC class I molecule is HLA-C*07:01. In one embodiment, the MHC class I molecule is HLA-C*07:02. In one embodiment, the MHC class I molecule is HLA-C*08:02. In one embodiment, the MHC class I molecule is HLA-C*12:03. In one embodiment, the MHC class I molecule is HLA-C*14:02.

[0083] In one embodiment, TAP (e.g., comprising or consisting of the sequences of SEQ ID NOs: 1-505 as defined herein) is non-covalently bonded to an MHC class I molecule (i.e., TAP is loaded into or non-covalently bonded to the peptide bond groove / pocket of the MHC class I molecule). In another embodiment, TAP is covalently bonded to an MHC class I molecule (alpha chain). In such constructs, TAP and the MHC class I molecule (alpha chain) are generated as a synthetic fusion protein having typically short (e.g., 5-20 residues, preferably about 8-12, e.g., 10) flexible linkers or spacers (e.g., polyglycine linkers). In another embodiment, the disclosure provides a nucleic acid encoding a fusion protein comprising TAP as defined herein fused to an MHC class I molecule (alpha chain). In one embodiment, the MHC class I molecule (alpha chain)-peptide complex is polymerized. Accordingly, in another aspect, the present disclosure provides multimers of MHC class I molecules loaded (commonly or non-commonly) with the TAP described herein. Such multimers may be conjugated to a tag, such as a fluorescent tag, that enables the detection of the multimer. Numerous strategies for the production of MHC multimers have been developed, including MHC dimers, tetramers, pentamers, octamers, and the like (as outlined in Bakker and Schumacher, Current Opinion in Immunology 2005, 17:428-433). MHC multimers are useful, for example, for the detection and purification of antigen-specific T cells. Accordingly, in another aspect, the present disclosure provides CD8 specific to the TAP as defined herein. + This method provides a method for detecting or purifying (isolating, concentrating) T lymphocytes, which involves contacting a cell population with multimers of MHC class I molecules loaded (co- or non-co-co-) with TAP, and CD8 conjugated by the MHC class I multimers. + This includes detecting or isolating T lymphocytes conjugated by MHC class I multimers. + T lymphocytes may be isolated using known methods, such as fluorescence-activated cell sorting (FACS) or magnetically activated cell sorting (MACS).

[0084] In another embodiment, the Disclosure provides cells (e.g., host cells) and, in one embodiment, provides isolated cells comprising the nucleic acids, vectors, or plasmids of the Disclosure (i.e., nucleic acids or vectors encoding one or more TAPs). In another embodiment, the Disclosure provides cells expressing, on their surface, an MHC class I molecule (e.g., one of the MHC class I molecules from the alleles disclosed above) bound to or presenting the TAP according to the Disclosure. In one embodiment, the host cell is a eukaryotic cell, e.g., a mammalian cell, preferably a human cell, cell line, or immortalized cell. In another embodiment, the cell is an antigen-presenting cell (APC). In one embodiment, the host cell is a primary cell, cell line, or immortalized cell. In another embodiment, the cell is an antigen-presenting cell (APC). The nucleic acids and vectors can be introduced into cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection” refer to techniques for introducing exogenous nucleic acids into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, and virus-mediated transfection. Preferred methods for transforming or transfecting host cells can be found, for example, in Sambrook et al. (above) and other laboratory manuals. Methods for introducing nucleic acids into mammalian cells in vivo are also known and may be used to target and deliver the vectors or plasmids of this disclosure for gene therapy.

[0085] Cells such as APCs can be loaded with one or more TAPs using various methods known in the art. As used herein, “loading cells with TAP” means that cells are transfected with RNA or DNA encoding TAP or TAP, or alternatively, that APCs are transformed with nucleic acids encoding TAP. Cells can also be loaded by contacting them with exogenous TAPs that can directly bind to MHC class I molecules present on the cell surface (e.g., cells pulsed with peptides). TAPs may also be fused to domains or motifs that facilitate their presentation by MHC class I molecules (e.g., endoplasmic reticulum (ER) retrieval signals, C-terminal Lys-Asp-Glu-Leu sequences (see Wang et al., Eur J Immunol. 2004 Dec;34(12):3582-94)).

[0086] In another embodiment, the Disclosure provides compositions or combinations / pools of peptides comprising any one or any combination of TAPs (or nucleic acids encoding such peptides) as defined herein. In one embodiment, a composition comprises any combination of TAPs as defined herein (any combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TAPs) or a combination of nucleic acids encoding such TAPs. Compositions comprising any combination / partial combination of TAPs as defined herein are encompassed in the Disclosure. In another embodiment, a combination or pool may comprise one or more known tumor antigens.

[0087] Accordingly, in another aspect, the present disclosure provides a composition comprising one or any combination of TAPs as defined herein (e.g., including or consisting of sequences SEQ ID NOs. 1 to 505 as defined herein) and a cell expressing an MHC class I molecule (e.g., one of the MHC class I molecules from the alleles disclosed above). The APCs for use in the present disclosure are not limited to any particular type of cell, but also include CD8 +This includes professional APCs such as dendritic cells (DCs), Langerhans cells, macrophages, and B cells, which are known to present proteinaceous antigens on their cell surface so as to be recognized by T lymphocytes. For example, APCs can be obtained by inducing DCs from peripheral blood monocytes and then contacting (stimulating) them with TAPs, either in vitro, ex vivo, or in vivo. APCs can also be activated to present TAPs in vivo, where one or more TAPs of this disclosure are administered to a subject, and APCs that present TAPs are induced in the subject's body. The phrases “inducing APCs” or “stimulating APCs” include contacting or loading cells with one or more TAPs or nucleic acids encoding TAPs, resulting in the presentation of TAPs on the surface of those cells by MHC class I molecules. As described herein, according to this disclosure, TAP may be indirectly loaded using, for example, a longer peptide / polypeptide (including a native protein) containing the sequence of TAP, which is then processed within the APC (e.g., by a protease) to generate a TAP / MHC class I complex on the cell surface. After loading the APC with TAP and causing the APC to present TAP, the APC can be administered to a subject as a vaccine. For example, ex vivo administration may include the steps of (a) collecting an APC from a first subject, (b) contacting / loading the APC from step (a) with TAP to form an MHC class I / TAP complex on the surface of the APC, and (c) administering the peptide-loaded APC to a second subject in need of treatment.

[0088] The first and second subjects may be the same subject (e.g., autovaccines) or different subjects (e.g., allovaccines). Alternatively, the Disclosure provides the use of the TAPs described herein (or combinations thereof) for producing compositions (e.g., pharmaceutical compositions) for inducing antigen-presenting cells. In addition, the Disclosure provides a method or process for producing pharmaceutical compositions for inducing antigen-presenting cells, the method or process comprising the step of mixing or compounding the TAPs, or combinations thereof, with a pharmaceutically acceptable carrier. Cells such as APCs expressing MHC class I molecules (e.g., any of the HLA molecules described above) loaded with any one or any combination of the TAPs defined herein are CD8 + T lymphocytes, for example, autologous CD8 + It may be used to stimulate / proliferate T lymphocytes. In other embodiments, the present disclosure may use any one or any combination of TAPs as defined herein (or nucleic acids or vectors encoding them), cells expressing MHC class I molecules, and T lymphocytes, more specifically CD8 + T lymphocytes (e.g., CD8) + The present invention provides a composition comprising a cell population including T lymphocytes.

[0089] In one embodiment, the composition further comprises a buffer, excipients, carriers, diluents, and / or a culture medium (e.g., a culture medium). In further embodiments, the buffer, excipients, carriers, diluents, and / or a culture medium are pharmaceutically acceptable buffers(s), excipients(s), carriers(s), diluents(s), and / or a culture medium(s). As used herein, “pharmaceutically acceptable buffers, excipients, carriers, diluents, and / or a culture medium” includes any and all solvents, buffers, binders, lubricants, fillers, thickeners, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizers, release retarders, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, etc., that are physiologically compatible, do not interfere with the effectiveness of the biological activity of the active ingredient(s), and are non-toxic to the subject. The use of such culture media and agents for pharmaceutically active substances is well known in the art (Rowe et al., Handbook of pharmaceutical excipients, 2003, 4 th (edition, Pharmaceutical Press, London UK). The use of any conventional culture medium or drug in the compositions of this disclosure is intended, except insofar as any conventional culture medium or drug is incompatible with the active compound (peptide, cell). In one embodiment, the buffer, excipient, carrier, and / or culture medium is a buffer, excipient, carrier, and / or culture medium that does not exist in nature. In one embodiment, one or more of the TAPs as defined herein, or nucleic acids (e.g., mRNA) encoding the above one or more TAPs, are contained in or complexed with lipid vesicles or liposomes, e.g., cationic liposomes (see, e.g., Vitor MT et al., Recent Pat Drug Deliv Formul. 2013 Aug;7(2):99-110) or other suitable carriers.

[0090] In another embodiment, the Disclosure provides a composition comprising one or any combination of TAPs as defined herein (e.g., comprising or consisting of sequences SEQ ID NOs. 1 to 505 as defined herein) (or nucleic acids such as mRNA encoding such peptides), as well as one or more of buffers, excipients, carriers, diluents, and / or culture media. For compositions comprising cells (e.g., APCs, T lymphocytes), the composition comprises a suitable culture medium that enables the maintenance of viable cells. Typical examples of such culture media include physiological saline, Earl's buffered salt solutions (Life Technologies®), or PlasmaLyte® (Baxter International®). In one embodiment, the composition (e.g., a pharmaceutical composition) is an “immunogenic composition,” a “vaccine composition,” or a “vaccine.” As used herein, the terms “immunogenic composition,” “vaccine composition,” or “vaccine” refer to a composition or formulation comprising one or more TAPs, nucleic acids, or vaccine vectors that, when administered to a subject, can induce an immune response to one or more TAPs present therein. Methods of vaccination for inducing an immune response in mammals (e.g., humans) include the use of a vaccine or vaccine vector administered by any conventional route known in the vaccine field, for example, via the mucosal surface (e.g., eye, nasal cavity, lung, oral, stomach, intestine, rectum, vagina, or urinary tract), via parenteral routes (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal), or by topical administration (e.g., via a transdermal delivery system such as a patch). In one embodiment, TAP (or a combination thereof) is conjugated to a carrier protein (conjugate vaccine) to increase the immunogenicity of TAP(plural). Accordingly, this disclosure provides a composition (conjugate) comprising TAP (or a combination thereof) or a nucleic acid (or a combination thereof) encoding TAP, and a carrier protein.For example, TAP(TAP) or nucleic acid(TAP) may be conjugated or complexed with a Toll-like receptor (TLR) ligand (see, e.g., Zom et al., Adv Immunol. 2012, 114:177-201), or a polymer / dendrimer (see, e.g., Liu et al., Biomacromolecules. 2013 Aug 12;14(8):2798-806), such as a polymer-conjugated TLR agonist (see, e.g., Lynn et al., Nature Biotechnology 33:1201-1210 (2015), Lynn et al., Nature Biotechnology 38:320-332 (2020)). In one embodiment, the immunogenic composition or vaccine further comprises an adjuvant. "Adjuvant" refers to a substance that, when added to an immunogenic agent such as an antigen (TAP, nucleic acid, and / or cell as described herein), nonspecifically enhances or strengthens the immune response to the drug in a host upon exposure to the mixture.Examples of adjuvants currently used in the vaccine field include: (1) mineral salts (aluminum salts such as aluminum phosphate and aluminum hydroxide, calcium phosphate gel), squalene; (2) oil-based adjuvants (oil emulsions and surfactant-based formulations, etc.), e.g., MF59 (microfluidized detergent stabilized oil-in-water emulsion), QS21 (purified saponin), AS02[SBAS2] (oil-in-water emulsion + MPL + QS-21); (3) particulate adjuvants, e.g., virosome (monolayer liposome vehicle incorporating influenza hemagglutinin), AS04 ([SBAS4] aluminum salt containing MPL), ISCOMS (saponin and lipid structural complex), polylactide coglycol (PLG); (4) microbial derivatives (natural and synthetic). Examples include (1) monophosphoryl lipid A (MPL), Detox (MPL + M. Phlei cell wall skeleton), AGP[RC-529] (synthetic acylated monosaccharide), DC_Chol (lipoid immunostimulant that can self-construct into liposomes), OM-174 (lipid A derivative), CpG motif (synthetic oligonucleotide containing an immunostimulatory CpG motif), modified CT and LT (genetically modified bacterial toxin immunohistogens to provide non-toxic adjuvant effects), (5) endogenous human immunomodulators, such as hGM-CSF or hIL-12 (cytokines that can be administered as either a protein or an encoded plasmid), Immudaptin (C3d tandem array), and / or (6) inert vehicles such as gold particles.

[0091] In one embodiment, TAP(or nucleic acids) (e.g., containing or consisting of sequences of sequence numbers 1 to 505 as defined herein) (or nucleic acids such as mRNA encoding the peptide(or)), or a composition containing the same, is in a lyophilized form. In another embodiment, TAP(or), nucleic acids(or), or a composition containing them is a liquid composition. In further embodiments, TAP(or) or nucleic acids(or) are present in the composition at concentrations of about 0.01 μg / mL to about 100 μg / mL. In further embodiments, TAP(or) or nucleic acids(or) are present in the composition at concentrations of about 0.2 μg / mL to about 50 μg / mL, about 0.5 μg / mL to about 10, 20, 30, 40, or 50 μg / mL, about 1 μg / mL to about 10 μg / mL, or about 2 μg / mL.

[0092] As described herein, cells such as APCs expressing MHC class I molecules loaded with or bound to any one or any combination of the TAPs defined herein may undergo CD8 in vivo or ex vivo. + It may be used to stimulate / amplify T lymphocytes. In another embodiment, the present disclosure provides a T cell receptor (TCR) molecule that can interact with or bind to the MHC class I molecule / TAP complex described herein, a nucleic acid molecule encoding such a TCR molecule, and a vector comprising such a nucleic acid molecule. The TCRs according to the present disclosure can specifically interact with or bind to TAP loaded onto or presented by an MHC class I molecule, preferably on the surface of a living cell in vitro or in vivo.

[0093] As used herein, the term TCR refers to an immunoglobulin superfamily member that has a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail, and can specifically bind to an antigen peptide bound to an MHC receptor (see, for example, Janeway et al, Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p.4:33, 1997). TCRs can be found on the surface of cells and generally consist of heterodimers having α and β chains (also known as TCRα and TCRβ, respectively). Similar to immunoglobulins, the extracellular portions of the TCR chain (e.g., α-chain, β-chain) contain two immunoglobulin regions, variable regions (e.g., TCR variable α-region or Vα, and TCR variable β-region or Vβ; typically amino acids 1-116 based on Rabat numbering at the N-terminus) and one constant region adjacent to the cell membrane (e.g., TCR constant domain α or Cα, typically amino acids 117-259 based on Rabat, and TCR constant domain β or Cβ, typically amino acids 117-295 based on Rabat). Also, similar to immunoglobulins, the variable domains contain complementarity-determining regions (CDRs, three per chain) separated by a framework region (FR). In certain embodiments, the TCR is found on the surface of T cells (or T lymphocytes) and associates with the CD3 complex.

[0094] By applying TCRs and, specifically, nucleic acids encoding the TCRs of this disclosure, for example, T lymphocytes (e.g., CD8) + T lymphocytes (e.g., CD8) or other types of lymphocytes that generate novel T lymphocyte clones that specifically recognize the MHC class I / TAP complex may be genetically transformed / modified. In certain embodiments, T lymphocytes obtained from a patient (e.g., CD8) may be used. + T lymphocytes are transformed to express one or more TCRs that recognize TAP, and the transformed cells are administered to the patient (autologous cell transfusion). In certain embodiments, T lymphocytes obtained from a donor (e.g., CD8) are used.+ T lymphocytes are transformed to express one or more TCRs that recognize TAP, and the transformed cells are administered to the recipient (allogeneic cell transfusion). In another embodiment, the present disclosure describes a T lymphocyte, e.g., CD8, transformed / transfected with a vector or plasmid encoding a TAP-specific TCR. + T lymphocytes are provided. In further embodiments, the disclosure provides a method for treating a patient with autologous or allogeneic cells transformed with a TAP-specific TCR. In certain embodiments, the TCR is expressed in primary T cells (e.g., cytotoxic T cells) by replacing an endogenous locus (e.g., endogenous TRAC and / or TRBC locus) using, for example, CRISPR, TALEN, zinc finger nuclease, or other targeted disruption systems.

[0095] In another embodiment, the disclosure provides a nucleic acid encoding the TCR described above. In a further embodiment, the nucleic acid resides in a vector such as the vector described above.

[0096] Further embodiments provide the use of tumor antigen-specific TCRs in the production of autologous or allogeneic cells for the treatment of cancers such as melanoma.

[0097] In some embodiments, patients treated with the compositions of the Disclosure (e.g., pharmaceutical compositions) are treated before or after treatment with antitumor agents and / or immunotherapies (e.g., CAR therapy, immune checkpoint inhibitor therapy). The compositions of the Disclosure include allogeneic T lymphocytes (e.g., CD8) that are ex vivo activated against TAP. + T lymphocytes), allogeneic or autologous APC vaccines loaded with TAP, vaccines containing nucleic acids (e.g., mRNA) encoding TAP, and allogeneic or autologous T lymphocytes (e.g., CD8 +This includes T lymphocytes, or lymphocytes transformed with a tumor antigen-specific TCR. The method for providing a T lymphocyte clone capable of recognizing TAP according to the present disclosure may be prepared for tumor cells expressing TAP in a subject (e.g., graft recipient), e.g., allogeneic T lymphocytes and / or donor lymphocyte infusion (DLI) recipients, and can be specifically targeted thereto. Accordingly, the present disclosure relates to a T cell receptor that can specifically recognize or bind to the TAP / MHC class I molecular complex and expresses CD8 + Provides T lymphocytes. These T lymphocytes (e.g., CD8 + CD8 (T lymphocytes) may be recombinant (manipulated) or naturally selected T lymphocytes. Therefore, this specification refers to CD8 + The present invention provides at least two methods for producing T lymphocytes, comprising the step of contacting undifferentiated lymphocytes with a TAP / MHC class I molecule complex (typically expressed on the surface of cells such as APCs) under conditions that induce T cell activation and T cell proliferation, which may be carried out in vitro or in vivo (i.e., in patients administered an APC vaccine in which APCs are loaded with TAP, or in patients treated with a TAP vaccine). By using a combination or pool of TAPs bound to MHC class I molecules, CD8s can recognize multiple TAPs. + It is possible to generate a population of T lymphocytes. Alternatively, tumor antigen-specific or targeted T lymphocytes can be MHC class I molecule / TAP complexes (i.e., engineered or recombinant CD8). +The TAP-specific TCR may be produced / generated in vitro or ex vivo by cloning one or more nucleic acids (genes) encoding a TCR (more specifically, α-chain and β-chain) that specifically binds to T lymphocytes. The nucleic acids encoding the TAP-specific TCR of this disclosure may be obtained ex vivo from T lymphocytes activated against TAP (e.g., by TAP-loaded APC) or from an individual exhibiting an immune response to a peptide / MHC molecular complex, using methods known in the art. The TAP-specific TCR of this disclosure may be recombinantly expressed in host cells and / or host lymphocytes obtained from a graft recipient or graft donor and optionally differentiated in vitro to provide cytotoxic T lymphocytes (CTLs). The nucleic acids (or more) (or more) encoding the TCR alpha-chain and β-chain may be introduced into T cells (e.g., from the subject being treated or from another individual) using any preferred method such as transfection (e.g., electroporation) or transduction (e.g., using a viral vector). Modified CD8 expressing a TCR specific to TAP + T lymphocytes may be proliferated in vitro using well-known culture methods.

[0098] This disclosure provides a method for producing immunoeffector cells expressing TCRs as described herein. In one embodiment, the method comprises transfecting or transfecting immunoeffector cells (e.g., immunoeffector cells isolated from a subject such as a subject having melanoma) so that the immunoeffector cells express one or more TCRs as described herein. In a particular embodiment, the immunoeffector cells are isolated from an organism and genetically modified without further in vitro manipulation. Such cells can then be directly re-administered to the organism. In a further embodiment, the immunoeffector cells are first activated and stimulated to proliferate in vitro and then genetically modified to express TCRs. In this regard, the immunoeffector cells may be cultured before or after genetic modification (i.e., transfected or transfected to express TCRs as described herein).

[0099] Prior to the in vitro manipulation or genetic modification of the immunoeffector cells described herein, the cell source may be obtained from a subject. In particular, immunoeffector cells for use with the TCR described herein include T cells. T cells can be obtained from several sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus issue, tissue from infection sites, ascites, pleural fluid, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from units of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL® isolation. In one embodiment, cells derived from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes and includes T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis may be washed to remove the plasma fraction, and the cells may be placed in a suitable buffer or culture medium for further processing. In one embodiment of the present invention, cells are washed with PBS. In alternative embodiments, the washing solution may be calcium-deficient, magnesium-deficient, or many but not all divalent cations. As will be understood by those skilled in the art, the washing step may be achieved by methods known to those skilled in the art, such as by using semi-automatic flow-through centrifugation. After washing, cells may be resuspended in various biocompatible buffers, or other salines with or without buffers. In certain embodiments, undesirable components of the apheresis sample may be removed in cells resuspended directly in culture medium. In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing erythrocytes and depleting monocytes (e.g., by centrifugation via a PERCOLL® gradient). CD28 + CD4 + CD8 + CD45RA + , and CD45RO +Specific subpopulations of T cells, such as 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 using a combination of antibodies targeting surface markers specific to negatively selected cells. One method for use herein is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a cocktail of monoclonal antibodies targeting cell surface markers present on negatively selected cells. For example, to enrich CD8+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD4. Alternatively, flow cytometry and cell sorting may be used to isolate the cell population of interest for use in this disclosure. PBMCs may be used directly for gene modification by TCR using the methods described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic T lymphocytes and helper T lymphocytes can be sorted into subpopulations of naive T cells, memory T cells, and effector T cells, either before or after gene modification and / or proliferation.

[0100] This disclosure relates to isolated immune cells, such as T lymphocytes (e.g., CD8), that are specifically induced, activated, and / or amplified (proliferated) by TAP (i.e., TAP bound to MHC class I molecules expressed on the surface of cells), or a combination of TAPs. + This disclosure also provides T lymphocytes. This disclosure also provides TAPs or combinations thereof as described herein (i.e., one or more TAPs bound to an MHC class I molecule) and CD8 cells that can recognize such TAPs. + A composition containing T lymphocytes is provided.

[0101] In another aspect, the disclosure relates to T lymphocytes (e.g., CD8) that specifically recognize one or more MHC class I molecule / TAP complexes as described herein.+ A concentrated cell population or cell culture (e.g., CD8) in T lymphocytes. + This provides a population of T lymphocytes. Such a concentrated population may be obtained by ex vivo proliferation of specific T lymphocytes (e.g., TILs) using cells such as APCs expressing MHC class I molecules loaded with one or more of the TAPs disclosed herein (e.g., presenting them). As used herein, “concentrated” refers to a population of tumor antigen-specific T lymphocytes (e.g., CD8). + This means that the proportion of T lymphocytes is significantly higher compared to the natural population of cells, i.e., compared to those not subjected to the step of ex vivo proliferation of specific T lymphocytes. In one embodiment, the cell population is a TIL population or derived from TILs, e.g., TILs isolated from a patient and TILs proliferated ex vivo. In a further embodiment, the cell population contains TAP-specific T lymphocytes (e.g., CD8 + The proportion of T lymphocytes is at least about 0.5%, for example, at least about 1%, 1.5%, 2%, or 3%. In some embodiments, TAP-specific T lymphocytes (e.g., CD8) are present in the cell population. + The proportion of T lymphocytes is approximately 0.5-10%, 0.5-8%, 0.5-5%, 0.5-4%, 0.5-3%, 1-5%, 1-4%, 1-3%, 2-5%, 2-4%, 2-3%, 3-5%, or 3-4%. Such cell populations or cultures (e.g., CD8 + A population of T lymphocytes that specifically recognize one or more target MHC class I molecule / peptide (TAP) complexes (e.g., CD8 + The concentrated T lymphocytes (e.g., CD8) may be used in tumor antigen-based cancer immunotherapy, as detailed below. In some embodiments, TAP-specific T lymphocytes (e.g., CD8) may be used. +Populations of T lymphocytes (e.g., CD8) are further enriched using affinity-based systems, such as multimers of MHC class I molecules (commonly or non-commonly) loaded with TAP(plural) as defined herein. Thus, this disclosure relates to TAP-specific T lymphocytes (e.g., CD8). + A purified or isolated population of T lymphocytes, for example, TAP-specific T lymphocytes (e.g., CD8 + The present invention provides purified or isolated populations in which the proportion of T lymphocytes is at least approximately 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0102] In another aspect, the present disclosure provides antibodies or antigen-binding fragments thereof (e.g., TCR mimic antibodies or TCR-like antibodies) or soluble TCRs, in which the TAP described herein specifically binds to complexes bound to HLA molecules, such as multiple HLA molecules as defined herein. As used herein, the term “antibody or antigen-binding fragment” refers to all types of antibodies / antibody fragments, including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, humanized antibodies, CDR-graft antibodies, chimeric antibodies, and antibody fragments, as long as they exhibit the desired antigen specificity / binding activity. An antibody fragment is a portion of a full-length antibody, generally comprising its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., single-chain Fv, scFv), single-domain antibodies (e.g., camelid-derived), shark NAR single-domain antibodies, and multispecific antibodies formed from antibody fragments, single-chain diabodies (scDb), bispecific T cell engagers (BiTE), biaffinity retargeting molecules (DART), bivalent scFv-Fc, and trivalent scFv-Fc. Antibody fragments are V H Area (V H , V H -V HThe term can also refer to binding sites containing CDRs or antigen-binding domains, such as antikalin, peptobodies, antibody-T cell epitope fusions (troybodies), or peptibodies. In one embodiment, the antibody or its antigen-binding fragment is a single-chain antibody, preferably a single-chain Fv (scFv). In one embodiment, the antibody or its antigen-binding fragment includes at least one constant domain, e.g., a constant domain of the light chain and / or heavy chain, or a fragment thereof. In a further embodiment, the antibody or its antigen-binding fragment includes a crystallizable (Fc) fragment of the constant heavy chain of the antibody. In one embodiment, the antibody or antigen-binding fragment is scFv (scFV-Fc) containing an Fc fragment. In one embodiment, the scFv component is connected to the Fc fragment by a linker, e.g., a hinge. The presence of the Fc region is useful for inducing a response to tumor cells by complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated phagocytosis (ADCP), or antibody-dependent cell-mediated cytotoxicity (ADCC).

[0103] In one embodiment, the antibody or its antigen-binding fragment is a multispecific antibody or its antigen-binding fragment, such as a bispecific antibody or its antigen-binding fragment, where at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognizes a complex containing the TAP described herein, bound to an HLA molecule. In one embodiment, at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognizes an immune cell effector molecule. The term "immune cell effector molecule" refers to a molecule (e.g., a protein) expressed by an immune cell whose engagement by the multispecific antibody or antibody fragment results in activation of the immune cell. Examples of immune cell effector molecules include the CD3 signaling complex in T cells, such as CD8 T cells, and various activating receptors on NK cells (e.g., NKG2D, KIR2DS, NKp44). In a further embodiment, at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognizes and engages with the CD3 signaling complex in a T cell (e.g., anti-CD3). In a further embodiment, the multispecific antibody or antibody fragment is a single-stranded diabody (scDb). In further embodiments, scDb comprises a first antibody fragment (e.g., scFv) that binds to a complex containing TAP as described herein, conjugated to an HLA molecule, and a second antibody fragment (e.g., scFv) that binds to and engages with an immune cell effector molecule, such as an intracellular CD3 signaling complex (e.g., anti-CD3 scFv). Such constructs may be used, for example, to induce cytotoxic T cell-mediated killing of tumor cells expressing a tumor antigen / MHC complex recognized by a multispecific antibody or antibody fragment. The antibody or its antigen-binding fragment may also be used as a chimeric antigen receptor (CAR) for producing CAR T cells, CAR NK cells, etc. A CAR combines a ligand-binding domain (e.g., antibody or antibody fragment) that provides specificity to a desired antigen (e.g., an MHC / TAP complex) with an activated intracellular domain (or signaling domain) moiety, such as a T cell or NK cell activation domain, to provide a primary activation signal.Antibodies that can bind to molecules expressed by tumor cells, more specifically the antigen-binding fragments of scFv, are commonly used as ligand-binding domains in CARs.

[0104] In one embodiment, the soluble TCR is a therapeutically soluble bispecific TCR (see, for example, Robinson et al., FEBS J. 2021 Nov;288(21):6159-6173, and Dilchert et al., Antibodies (Basel). 2022 May 10;11(2):34).

[0105] In one embodiment, the TCR or soluble TCR comprises a TCR beta (β) chain containing a complementarity-determining region 3 (CDR3) which includes one of the amino acid sequences defined in Table 3 (right column).

[0106] In one embodiment, a soluble TCR, antibody, or antibody fragment (e.g., a TCR-mimicking antibody) binds to an antitumor agent to form an antibody-drug conjugate (ADC). Such an ADC enables the delivery of an antitumor agent to tumor cells expressing one or more TAPs as described herein (see, for example, Shen et al., Asian J Pharm Sci. 2020 Nov;15(6):777-785).

[0107] This disclosure also provides nucleic acids such as soluble TCRs, antibodies, antibody fragments, or mRNA encoding CARs as described herein. Such nucleic acids may be formulated into suitable vehicles such as lipid nanoparticles, as described above, and may be used in the treatment of cancers such as melanoma, as described below.

[0108] Therefore, in another aspect, the present disclosure provides host cells, preferably immune cells such as T cells or NK cells (i.e., CAR T cells or NK cells), that express an antibody or antibody fragment (e.g., scFv) described herein.

[0109] This disclosure relates to the aforementioned immune cells (CD8 + T lymphocytes, CAR T cells, CAR NK cells) or TAP-specific CD8 + The present invention further relates to a pharmaceutical composition or vaccine comprising a population of T lymphocytes. Such a pharmaceutical composition or vaccine may comprise one or more pharmaceutically acceptable excipients and / or adjuvants as described above.

[0110] In another aspect, the Disclosure further relates to the use of any TAP, nucleic acid, expression vector, T cell receptor, antibody / antibody fragment, cell (e.g., T lymphocyte, APC, CAR T cell, CAR NK cell), and / or composition, or any combination thereof, comprising or relating to any of the sequences of Sequence ID No. 1 to 505 as defined herein, as a pharmaceutical for the treatment of cancer (e.g., melanoma), or in the manufacture of a pharmaceutical. The Disclosure further relates to any TAP, nucleic acid, expression vector, T cell receptor, antibody / antibody fragment, cell (e.g., T lymphocyte, APC), and / or composition (e.g., vaccine composition), or any combination thereof, for use, for example, as a melanoma cancer vaccine in the treatment of cancer (e.g., melanoma). The TAP sequences identified herein may be used for the production of synthetic peptides, i) used for in vitro stimulation and proliferation of tumor antigen-specific T cells injected into tumor patients, and / or ii) used as a vaccine to induce or enhance an antitumor T cell response in cancer (e.g., melanoma) patients.

[0111] In another embodiment, the Disclosure provides the use of TAP described herein (e.g., comprising or consisting of any of the sequences of Sequence ID No. 1 to 505 as defined herein), or a combination thereof (e.g., a peptide pool), or one or more nucleic acids encoding TAP(s) for use as a vaccine for treating cancer (e.g., melanoma) in a subject. The Disclosure also provides TAP described herein, or a combination thereof (e.g., a peptide pool), or one or more nucleic acids encoding TAP(s) for use as a vaccine for treating cancer (e.g., melanoma) in a subject. In one embodiment, the subject is TAP-specific T lymphocytes (e.g., CD8 + The recipient is a T lymphocyte. In another aspect, the present disclosure provides a method for treating cancer, more specifically melanoma (e.g., reducing the number of tumor cells, killing tumor cells), the method providing to a target requiring it an effective amount of one or more MHC class I molecule / TAP complexes (expressed on the surface of cells such as APCs) that recognize (i.e., express TCRs that bind to them) T lymphocytes (e.g., CD8 + This method includes administering (injecting) T lymphocytes. In one embodiment, this method involves the CD8 + The method further comprises administering to the subject an effective amount of TAP or a combination thereof, or one or more nucleic acids encoding TAP and / or cells expressing MHC class I molecules (e.g., APCs such as dendritic cells) loaded with TAP(-) after administration / infusion of T lymphocytes. In further embodiments, the method comprises administering to the subject in need a therapeutically effective amount of dendritic cells loaded with one or more TAP. In further embodiments, the method comprises administering to the patient in need allogeneic or autologous cells expressing recombinant TCRs that bind to TAP presented by MHC class I molecules.

[0112] In another aspect, the Disclosure relates to T lymphocytes (e.g., CD8) that recognize one or more MHC class I molecules loaded (presented) with TAP for the treatment of cancer, more specifically melanoma (e.g., reducing the number of tumor cells, killing tumor cells). + In another aspect, the disclosure provides the use of T lymphocytes (e.g., CD8) that recognize one or more MHC class I molecules loaded (presented) with TAP for the preparation / manufacturing of pharmaceuticals for treating cancer, more specifically melanoma (e.g., reducing the number of tumor cells, killing tumor cells). + In another aspect, the disclosure provides the use of T lymphocytes (e.g., CD8) that recognize one or more MHC class I molecules loaded (presented) with TAP for use in the treatment of cancer, more specifically melanoma (e.g., to reduce the number of tumor cells, to kill tumor cells). + The present invention provides T lymphocytes, or combinations thereof. In a further embodiment, use further comprises the use of cells (e.g., APCs) expressing an effective amount of TAP (or a combination thereof), or one or more nucleic acids encoding TAP(or more), and / or one or more MHC class I molecules loaded (presented) with TAP, after the use of the TAP-specific T lymphocytes.

[0113] This disclosure also provides a method for generating an immune response against tumor cells (e.g., melanoma cells) expressing human class I MHC molecules loaded with any of the TAPs disclosed herein (e.g., any of the sequences of SEQ ID NOs. 1 to 505 as defined herein) or a combination thereof, the method comprising administering cytotoxic T lymphocytes that specifically recognize class I MHC molecules loaded with the TAP or a combination of TAPs. This disclosure also provides the use of cytotoxic T lymphocytes that specifically recognize class I MHC molecules loaded with any of the TAPs disclosed herein or a combination of TAPs to generate an immune response against tumor cells expressing human class I MHC molecules loaded with the TAP or a combination of TAPs.

[0114] TAP, its combinations (e.g., peptide pools), nucleic acids (or multiple entities) encoding TAP(or entities), and antibodies, TCRs, cells (e.g., CD8 T cells, APCs), vaccines, and compositions disclosed herein may be used for the prevention or treatment of any cancer expressing TAP, such as melanoma.

[0115] The TAPs, combinations thereof (e.g., peptide pools), nucleic acids (or multiple) encoding TAPs, as well as antibodies / antibody fragments, TCRs, cells (e.g., T lymphocytes, CAR T cells, or NK cells, APCs), vaccines, and compositions disclosed herein may be used to induce or stimulate an immune response against cancer cells expressing TAPs. In one embodiment, the cancer is melanoma. In one embodiment, the melanoma is cutaneous melanoma. In one embodiment, the melanoma is a superficial spreading subtype. In one embodiment, the melanoma is a nodular subtype. In one embodiment, the melanoma is a lentiginous malignant subtype. In one embodiment, the melanoma is a nodular subtype. In one embodiment, the melanoma is an acral lentiginous subtype. In one embodiment, the melanoma is ocular melanoma (e.g., choroidal melanoma, iris melanoma, or uveal melanoma). In one embodiment, the melanoma is mucosal melanoma. In one embodiment, the melanoma is stage 0 melanoma. In one embodiment, the melanoma is stage 1 melanoma. In one embodiment, the melanoma is stage 2 melanoma. In one embodiment, the melanoma is stage 3 melanoma. In one embodiment, the melanoma is stage 4 melanoma. In one embodiment, the melanoma is recurrent melanoma. In one embodiment, the melanoma is chemotherapy-resistant melanoma.

[0116] In one embodiment, the method or use described herein further comprises determining the HLA class I alleles expressed by the patient prior to treatment / use, and administering or using a TAP that binds to one or more of the HLA class I alleles expressed by the patient. For example, if a patient is determined to express HLA-01*01 and HLA-B40*01, any combination of (i) TAP (binding to HLA-A01*01) of SEQ ID NOs: 11, 14, 20, 27, 29, 37, 86, 87, 121, 150, 165, 173, 279, 288, 299, 308, 312, 316, 319, 331, 340, 352, 362, 363, 384, 395, 398, 411, 419, 429, 458, 474, 476, 487, 492, 500, and / or 501 and (ii) SEQ ID NOs: 215 and / or 322 (binding to HLA-B40*01) may be administered to or used in the patient.

[0117] In some embodiments, patients treated with the compositions of the Disclosure (e.g., pharmaceutical compositions) are treated before or after treatment with allogeneic stem cell transplantation (ASCL), allogeneic lymphocyte infusion, or autologous lymphocyte infusion.

[0118] In one embodiment, the TAP, nucleic acids, expression vectors, T cell receptors, antibodies / antibody fragments, cells (e.g., T lymphocytes, CAR T cells or NK cells, APCs), and / or compositions, or any combination thereof, according to the Disclosure, may be one or more additional activators or therapies for treating melanoma, e.g., chemotherapy (e.g., vinca alkaloids, agents that inhibit microtubule formation (e.g., colchicine and its derivatives), monomethyl auristatin E (MMAE)), anti-angiogenic agents, therapeutic antibodies, EGFR targeters, tyrosine kinase targeters (e.g., tyrosine kinase inhibitors), transition metal complexes, proteasome inhibitors, antimetabolites (e.g., nucleoside analogs), alkylating agents, platinum-based drugs, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids (e.g., all-trans retinoic acid) It may be used in combination with: or derivatives thereof), geldanamycin or its derivatives (e.g., 17-AAG), inhibitors of CDK4 / 6, TGF-β, WNT-β-catenin, MYC, or PI3K, surgery, immune checkpoint inhibitors or immunotherapies (e.g., PD-1 / PD-L1 inhibitors such as anti-PD-1 / PD-L1 antibodies, CTLA-4 inhibitors such as anti-CTLA-4 antibodies, B7-1 / B7-2 inhibitors such as anti-B7-1 / B7-2 antibodies, TIM3 inhibitors such as anti-TIM3 antibodies, BTLA inhibitors such as anti-BTLA antibodies, CD47 inhibitors such as anti-CD47 antibodies, GITR inhibitors such as anti-GITR antibodies), antibodies against tumor antigens (e.g., anti-CD19, anti-CD22 antibodies), cell-based therapies (e.g., CAR T cells, CAR NK cells), and cytokines such as IL-2, IL-7, IL-21, and IL-15. In one embodiment, the TAP, nucleic acids, expression vectors, T cell receptors, cells (e.g., T lymphocytes, APCs), and / or compositions according to the Disclosure are administered / used in combination with immune checkpoint inhibitors. In one embodiment, the TAP, nucleic acids, expression vectors, T cell receptors, cells (e.g., T lymphocytes, APCs), and / or compositions according to the Disclosure are administered / used in combination with radiotherapy.In one embodiment, the TAP, nucleic acids, expression vectors, T cell receptors, antibodies / antibody fragments, cells (e.g., T lymphocytes, CAR T cells or NK cells, APCs), and / or compositions according to the Disclosure are administered / used in combination with surgery. In one embodiment, the TAP, nucleic acids, expression vectors, T cell receptors, antibodies / antibody fragments, cells (e.g., T lymphocytes, CAR T cells or NK cells, APCs), and / or compositions according to the Disclosure are administered / used in combination with inhibitors of CDK4 / 6, TGF-β, and / or WNT-β-catenin. Several CDK4 / 6 inhibitors include Palbociclib (PD-0332991, Ibrance), Ribociclib (LEE-011, Kisqali), Abemaciclib (LY2835219, Verzenios), SHR6390, and Trilaciclib (G1T28), which are in clinical trials. Inhibitors of TGF-β include antisense inhibitors such as AP12009 (Trabedersen) and ISTH0036, antibodies and ligand traps such as GC1008 (Fresolimumab), LY2382770, and P144, vaccines targeting the TGF-β pathway such as Belagenpumatucel-L (Lucanix®), and small molecule inhibitors such as FANG® or Vigil (Gemogenovatucel-T), LY2157299 (Galunisertib), and TEW-7197. Inhibitors of the WNT-β-catenin pathway include amino acid starvation factors (asparaginase), GSK3 inhibitors, and C2. [ka] Examples include WNT974, ETC-1922159, RXC004, CGX1321, OTSA101-DTPA-90Y, Vantictumab (OMP-18R5), Ipafricept (OMP-54F28), PRI-724, SM08502, secreted frizzled-related proteins / peptides, and tankyrase inhibitors (XAV939, JW-55, RK-287107, and G007-LK).

[0119] Additional therapies may be administered before, concurrently with, or after administration of TAP, nucleic acids, expression vectors, T cell receptors, antibodies / antibody fragments, cells (e.g., T lymphocytes, CAR T cells or NK cells, APCs), and / or compositions as disclosed herein. [Examples]

[0120] This disclosure is illustrated in further detail by the following non-limiting embodiments.

[0121] Example 1: Identification of candidate melanoma tumor antigens Sample acquisition. The 12 rapidly frozen cutaneous melanoma samples used in this study were purchased from Tissue Solutions. This project was approved by the Research Ethics Boards of the Universite de Montreal. 700 mg to 1.4 g per tumor were used for mass spectrometry, and 30 mg to 70 mg were used for RNA sequencing. Chong et al. 1 Immunopeptome analysis data and RNA sequencing data from melanoma cell lines derived from seven patients were downloaded from the European Genome-phenome Archive (EGA) with access number EGAS00001003724.

[0122] Immunoprecipitation of MHC I. W6 / 32 antibody (BioXcell) was incubated in PBS at room temperature for 60 minutes using PureProteome® protein A magnetic beads (Millipore) at a ratio of 1 mg of antibody per 1 mL of slurry. As previously described, the antibody was covalently crosslinked to the magnetic beads using dimethyl pimeridate. 2The beads were stored at 4°C in PBS at pH 7.2 and 0.02% NaN3. Primary tumor tissue samples were cut into small pieces (cubes, approximately 3 mm in size) and 6 ml of ice-cold PBS containing a protein inhibitor cocktail (Sigma, catalog no. P8340-5 ml) was added. The tissue was homogenized twice for 20 seconds using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set to 20,000 rpm. Then, 700 μl of ice-cold 10-fold lysis buffer (5% w / v CHAPS) was added to each sample. After incubation at 4°C for 60 minutes with tumbling, the tissue samples were centrifuged at 16,000 g at 4°C for 20 minutes. The supernatant was transferred to a new tube containing 1 mg of W6 / 32 antibody covalently crosslinked protein A magnetic beads. The samples were incubated by tumbling at 4°C for 20 hours and then placed on a magnet to recover the MHC I complex bound to the magnetic beads. The magnetic beads were washed first with 8 × 1 mL of PBS, then with 1 × 1 mL of 0.1X PBS, and finally with 1 × 1 mL of water. The MHC I complex was eluted from the magnetic beads by acid treatment with 1% trifluoroacetic acid (TFA). To remove any remaining magnetic beads, the eluate was transferred to a 2 mL Costar mL Spin-X centrifuge filter (0.45 μm, Corning) and centrifuged at 1500 g for 5 minutes. Using a homemade stage tip packed with two 1 mm diameter octadecyl (C-18) solid-phase extraction disks (EMPORE), the peptide-containing filtrate was separated from the MHC I subunits (HLA molecules and β-2 macroglobulin). The stage tip was pre-washed first with methanol, then with 80% acetonitrile (ACN) in 0.1% TFA, then with 0.1% TFA, and finally with 1% TFA. The sample was loaded onto the stage tip, and the peptide was retained on the stage tip while HLA molecules and β-2 macroglobulin were found in the flow-through. The stage tip was washed with 1% TFA, then with 0.1% TFA, and the peptide was eluted in 30% ACN in 0.1% TFA. The peptide was dried using vacuum centrifugation and then stored at -20°C until MS analysis.

[0123] TMT labeling. Thumb-labeled TMT was performed on 12 primary melanoma samples used in the study. Samples were reconstituted in 20 μL of 200 mM HEPES buffer (pH 8.2). TMT reagent (Thermo Fisher Scientific) was dissolved in 40 μL of anhydrous ACN (Sigma-Aldrich), and 50 μg or 100 μg of the reagent was added to the peptide. The solution was gently mixed and incubated at room temperature for 90 minutes without agitation, after which the reaction was quenched with hydroxylamine (Thermo Fisher Scientific). Samples were desalted on a Silica C18 UltraMicroSpin™ column (The Nest Group), dried, and reconstituted in 4% FA (EMD Millipore).

[0124] Liquid chromatography-tandem MS analysis. Dried peptide extracts were resuspended in 4% FA and loaded onto a homemade C18 analytical column (20 cm × 150 μm inner diameter packed with C18 Jupiter Phenomenex) on an EASY-nLC II system using a gradient from 0% to 30% ACN (0.2% FA) for 106 minutes and a flow rate of 600 nL / min. Two samples were analyzed with Q-Exactive HF and ten samples with Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). For Q-Exactive HF, analysis was performed in cation mode using a 1.6 kV Nanospray 2 source. Each complete MS spectrum acquired at 60,000 resolution was followed by 20 MS / MS spectra, 2 × 10⁶ at 60,000 resolution. 4 An automated gain-controlled target, an 800 ms injection time, and the most abundant polyvalent ions were selected for MS / MS sequencing with a collision energy of 28%. Analysis using an Orbitrap Fusion mass spectrometer was performed in cation mode with a 2.8 kV Nanoflex source. Each complete MS spectrum acquired at a resolution of 120,000 was followed by 20 MS / MS spectra at a resolution of 50,000, 2 × 10⁻¹⁶. 4We selected the most abundant polyvalent ions for MS / MS sequencing with an automated gain control target, an injection time of 1000 ms, and a collision energy of 35%.

[0125] Construction of an MS database. As described, k-mer profiling was used to assemble a cancer-specific proteome. 3、4 From each cancer sample database, k-mers (33 nucleotides in length) that appeared more than twice in the mTECs k-mer database were removed, and the remaining k-mers were assembled into contigs. Finally, the contigs were three-frame translated, and different polypeptides were linked using a "JJ" linker. Each of these databases was linked to the standard proteome of the corresponding sample and used for TA identification.

[0126] MAP identification. LC-MS / MS data was searched against relevant databases using Peaks X Pro (Bioinformatics Solution Inc.). For peptide identification, the tolerances for precursor and fragment ions were set to 10 ppm and 0.01 Da, respectively. Oxidation (M) and deamidation were set as variable modifications. In addition, for TMT labeling, the generation of K and N terms was set as a fixed modification, and the generation of STY was set as a variable modification. After peptide identification, the modified target-decoy method built into PEAKS was used to apply a sample-specific threshold to the PEAKS score, ensuring a false detection rate (FDR) of 5%, calculated as the ratio between the number of decoy hits and the number of target hits exceeding the score threshold. A PEAKS score corresponding to a 5% FDR was determined for each sample, and peptides exceeding the threshold were further filtered to meet the following criteria: the peptide length was 8-11 amino acids, and the predicted likelihood rank of the elution ligand to any of the sample's HLA alleles based on NetMHCpan-4.1b was <2%. 5 These filtering steps were performed using MAPDP. 6 .

[0127] Selection of TSA, TAA, and LSA. TA candidates were selected as previously described, based on RNA expression of their sources in the origin cancer sample and mTECs (FC≧10). 3、4 Next, the expression of the coding sequences of candidate TAs was investigated using normal tissues from GTEx (n=11, up to 50 samples per tissue, 50 tissues), mTECs (n=11), purified blood and bone marrow samples (n=115), purified melanocytes (n=11), melanoma samples from TCGA (n=150, TCGA_SKCM), and Chong et al. 1 Melanoma cell lines (n=7) were evaluated using BamQuery. 4、7 The TAs listed herein are peptides that meet the following criteria: 1. mTSA originates from a variant genomic sequence supported by at least 5 reads and 5% of reads at a locus, and the variant is not reported in dbSNP version 155 (unless annotated as "pathogenic"). 2. In the case of non-mutant TA, the 95th percentile RNA expression level in TCGA_SKCM is at least twice as high as the 95th percentile RNA expression level in GTEx (excluding testicular and skin) and mTEC samples. A) In the case of aeTSA, the RNA of the aeTSA source is expressed at less than 8.55 reads per 100 million in over 90% of normal samples from mTECs, melanocytes, blood and bone marrow cells, and each GTEx tissue except testes. The mean expression is at least twice as high in TCGA_SKCM compared to normal GTEx skin, and the mean expression is at least twice as high in melanoma cell lines compared to purified melanocytes. B) In the case of TAA, the RNA of the TAA source can be expressed at more than 8.55 reads per 100 million parts (rphm) in more than 10% of samples from any normal tissue (GTEx, melanocytes, mTECs, and / or blood and bone marrow cells), but the mean expression is at least twice as high in TCGA_SKCM compared to each GTEx tissue except mTECs and testes, and the mean expression in melanoma cell lines is at least twice as high compared to purified melanocytes. C) In the case of LSA, the RNA of the LSA source is expressed at more than 8.55 rphm in at least 10% of GTEx skin samples or purified melanocytes, the mean expression in GTEx skin is higher compared to all other GTEx tissues, and is expressed at less than 8.55 rphm in more than 90% of normal samples from each GTEx tissue except mTEC, blood and bone marrow cells, as well as testes and skin.

[0128] TCGA analysis. TCGA RNA-seq gene expression data for hg38 were obtained as upper quartile-normalized fragments (FPKM-UQ) per kilobase of transcripts per million map reads using the R package TCGAbiolinks. For genes with duplicate entries, the mean expression across entries was calculated. The FPKM-UQ values ​​were then correlated with RPHM expression for each TA from BamQuery, or using the R package GSVA, as in Miranda et al. 18This was used to perform single-sample gene set enrichment analysis (ssGSEA) on the stem cell gene set reported by [research organization name]. Level 3 methylation data (HM450) processed from the liftover pipeline for TCGA samples had been previously obtained using the TCGAbiolinks package. Only probes within 2kb of the transcription start site of a given TA source gene were retained. Mean beta values ​​were used for genes associated with multiple probes. For local CNV correlations, copy number scores at the processed hg38 gene level had been previously obtained using the TCGAbiolinks package. Mean DNA copy numbers were used for multiple segments associated with the TA coding region. Mutation rate data for TCGA samples were obtained from Firebrowse (http: / / firebrowse.org / ) as the number of non-synonymous mutations per nucleotide (rate_non column). All correlations with TA RPHM expression were performed using the rcorr function in R. P values ​​were adjusted using the Benjamini-Hochberg method.

[0129] The characteristics of TSA, TAA, and LSA as identified herein are described in Tables 2A-2C. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 4-1 Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 4-6 Table 4-7 Table 4-8 Table 4-9 Table 4-10 Table 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]

[0130] Example 2: Evidence that aeTSA contributes to ICB response method Predicted TA presentation. HLA alleles of melanoma samples from previous studies on ICB were estimated from RNA-seq using Optitype. Indiscriminate binders for a given MAP (all HLA alleles capable of presenting the MAP) were obtained using NetMHCpan-4.1b. These corresponded to HLA alleles for which the given MAP had a predicted eluting ligand likelihood rank <2%. A given TA was considered presented in the sample if it had expression >0 RPHM and at least one of the patient's HLA allotypes was a potential binder. If a patient expressed two or more HLA alleles capable of presenting a TA, the TA was counted multiple times. Only cutaneous melanoma samples from previous studies were analyzed.

[0131] Riaz et al 8 Number of T cell clones proliferated during therapy in patients from Riaz et al. 8 TCRB chronotypes were downloaded from (https: / / www.github.com / riazn / bms038_analysis). Next, the FEST web tool (www.stat-apps.onc.jhmi.edu / FEST) was used to determine patient-specific proliferated TCRB clones with the following parameters: 1) pre-therapy sample as reference, 2) nTemplates_threshold=1, 3) Ignore_baseline_threshold=TRUE, 4) Nucleotide_level=FALSE, 5) FDR5% and OR5.

[0132] result Despite their high prevalence in cancer cells, little is known about the role of non-mutant TAs in spontaneous or treatment-induced antitumor immunity in vivo. To assess this, we predicted the number of non-mutant TAs in pretreatment biopsies from melanoma patients treated with ICBs (anti-PD-1, anti-PD-L1, and / or anti-CTLA). The presence of TAs in individual tumors was estimated based on expression based on two criteria: TA expression at the RNA level and homologous HLA allotype (i.e., presence of TA-HLA pairs). 4 In all datasets tested, patients presented a large number of TA-HLA pairs. Nevertheless, there was no significant difference in the number of TA-HLA pairs in pre-treatment samples between responders and non-responders (Figure 1A, Figure 2A, Figure 2B). Recent meta-analyses suggest that the lack of correlation between TA loading and response to ICB is generally due to a threshold effect. Having more TAs does not provide any advantage when one or some TAs are immunogenic. 10 Currently, the lack of immunopeptide analysis data and a complete set of non-mutant TAs from these patients prevents us from drawing definitive conclusions regarding the value of non-mutant TAs in predicting the response to ICB.

[0133] In a previous study of anti-PD-1 therapy in melanoma, Riaz et al. reported a linear correlation between the number of proliferating T cell clones and the number of mTSAs that were lost during therapy in responders (patients with partial or complete response, PRCR) but not in non-responders (patients with stable or progressive disease, SD or PD). 8 In particular, the data from Riaz et al. 8 Using this method, it was found that responders, rather than non-responders, showed a significant decrease in the number of non-mutant TA-HLA pairs during therapy (Figure 1B, Figure 2C, Figure 2D), which could not be explained by the decrease in tumor purity alone (Figure 2E). A positive correlation between the disappearance of non-mutant TAs and the response to ICB was validated in one of two additional datasets using pre-ICB and ICB samples. 11、12(Figure 2C, Figure 2D). Furthermore, the loss of the number of aeTSA-HLA in responders had a strong linear correlation with the number of T cell clones that proliferated during the therapy. On the other hand, for TAA and LSA, a positive but not significant correlation with T cell proliferation was observed (Figure 1C).

[0134] To determine whether the loss of aeTSA in responders was directly due to their recognition and elimination by specific T cell clonotypes, experiments were conducted to identify the cognate antigens of the proliferated T cell receptor Vβ (TCRB) sequences. For this purpose, the proliferated TCRB sequences from Riaz et al. 8 were first checked for overlap with a database of aeTSA-specific CD8 TCRB clonotypes obtained using the functional expansion of specific T cells (FEST) assay in previous studies 9 . In the FEST assay, TCRB sequencing is performed on T cells isolated from healthy donor PBMCs and stimulated or not stimulated with individual synthetic aeTSAs. Next, TCR clonotypes responsive to aeTSA are identified based on their significant proliferation in the aeTSA-stimulated state compared to controls. Surprisingly, one TCRB clone amplified in responders from Riaz et al. 4、15、16 was found to be specific for an exon aeTSA derived from cancer fetal IGF2BP1, which was previously identified in ovarian cancer 8 . The proliferation of this CD8 T cell clonotype was associated with the complete loss during aeTSA therapy from IGF2BP1, strongly indicating direct recognition and elimination (Figure 1D). 16 、cancer fetal IGF2BP1 17 was found to be specific for an exon aeTSA derived from cancer fetal IGF2BP1, which was previously identified in ovarian cancer

[0135] Example 3: Immunogenicity of Selected Melanoma Tumor Antigen Candidates The functional expansion of specific T cells (FEST) assay was performed on the melanoma aeTSAs identified herein.

[0136] Method Selection of aeTSAs for immunogenicity assays. Melanoma aeTSAs to be tested in a T cell-based immunogenicity assay were selected based on the following criteria. 1) Complete loss of expression during therapy (RPHM) in at least one responder from the cohort of Riaz et al. 8 with a rank elution < 2% for at least one HLA allele in NetMHCpan4.1b 5 ), 2) increased mean expression in TCGA-SKCM, 3) HLA allotypes presenting the aeTSA (HLA-A*02:01 or HLA-B*68:01 shared by melanoma samples and PBMC donors), 4) aeTSA biotype (25% standard, 75% non-standard), 5) novel MAP status.

[0137] Functional expansion of peptide-specific T cells (FEST). With minor modifications, T cells were cultured as described above 9 . Briefly, on day 0, thawed PBMC from D36 (Miltenyi Biotec) were enriched for T cells using a human pan T cell isolation kit (Miltenyi Biotec). T cells were resuspended in AIM V medium supplemented with 50 μg / mL gentamicin (ThermoFisher Scientific) and 1% HEPES at 2×10 6 / mL. The T cell negative fraction was irradiated with 30Gγ, washed, and resuspended in AIM V medium supplemented with 50 μg / mL gentamicin and 1% HEPES at 2×10 6The cells were resuspended at / mL. 2.5 ml of T cells and irradiated T-cell depleted cells were added to a 12-well plate, either with each peptide (GLS Biochem) (final concentration of 1 μg / mL) or without the peptide. The cells were cultured at 37°C in 5% CO2 for 10 days. On days 3 and 7, half of the culture medium was replaced with fresh medium containing 100 IU / mL IL-2, 50 ng / mL IL-7, and 50 ng / mL IL-15 (day 3) and 200 IU / mL IL-2, 50 ng / mL IL-7, and 50 ng / mL IL-15 (day 7). On day 10, a new batch of T-cell depleted cells was generated using thawed PBMCs from the same donor. These cells were irradiated with 30Gγ for 2 hours at 37°C, either with or without the peptide, at 1 μg / mL of the relevant peptide. These cells were washed and added to the culture in a 1:1 T cell:non-T cell ratio. On days 13 and 17, at least half of the culture medium was replaced with fresh medium (final concentrations: 100 IU / mL IL-2, 25 ng / mL IL-7, and 25 ng / mL IL-15). On day 20, cells were harvested and performed a FEST assay.

[0138] Functional proliferation (FEST) assay of specific T cells. Regarding the FEST assay, human CD8 + Using a T cell isolation kit (Miltenyi Biotec), CD8 + The cells were further isolated. CD8 was used as a negative control. + T cells were also isolated from freshly thawed, uncultured PBMCs of the same healthy donor. Using the QIAGEN DNA blood mini-kit (QIAGEN), CD8 + DNA was extracted from T cells. TCRVβ CDR3 sequencing was performed using the ultradeep resolution of the ImmunoSEQ platform (Adaptive Biotechnologies). Raw data exported from the immunoSEQ portal was processed using the FEST web tool. 9It was processed at (www.stat-apps.onc.jhmi.edu / FEST).

[0139] Results The ability of 12 selected aeTSAs to stimulate the proliferation of specific T cell clonotypes in vitro was evaluated by the FEST assay. The aeTSAs were able to induce specific T cell proliferation (Figure 3). The TCR β clonotypes expanded by each aeTSA are shown in Table 3.

Table 6-1

Table 6-2

Table 6-3

Table 6-4

[0140] These results indicate that the representative aeTSAs tested induce the proliferation of polyclonal T cells after in vitro stimulation, providing evidence that they may be useful for immunotherapy against melanoma.

[0141] The TCR recognition tendencies of the 200 representative aeTSAs and 5 mTSAs described herein were also evaluated using the PRedictor of Immunogenic Epitope PRIME2.0 web tool (http: / / ec2-18-188-210-66.us-east-2.compute.amazonaws.com:3000, Gfeller, D., et al., Improved predictions of antigen presentation and TCR recognition with MixMHCpred2.2 and PRIME2.0 reveal potent SARS-CoV-2 CD8+ T-cell epitopes. Cell Systems, 2023.14(1):p.72-83.e5). PRIME2.0 is based on a neural network and uses (1) predicted HLA-I presentation score (MixMHCpred2.2 -log(% rank)), (2) amino acid frequency at a position with minimal impact on HLA-I binding and a higher likelihood of facing the TCR, and (3) peptide length as input features. The PRIME2.0 scores of aeTSA and mTSA were compared to the scores of 590 known immunogenic peptides (53 HLA alleles that bind) and 5887 non-immunogenic peptides (62 HLA alleles that bind) used to train PRIME2.0. The results reported in Figure 3B show that the tested aeTSA has a PRIME2.0 score that is (1) equivalent to (and even better than) mTSA, (2) equivalent to the set of known immunogenic control peptides (neoantigens) used to train the PRIME2.0 algorithm, and (3) significantly higher than the set of non-immunogenic control peptides used to train the PRIME2.0 algorithm. The aeTSA of Sequence ID No. 1 described herein was shown to have a very high PRIME2.0 immunogenicity score of 0.31991. This data provides further evidence that the aeTSA described herein is immunogenic and may be useful in immunotherapy for melanoma.

[0142] Example 4: Further evidence that aeTSA is a good candidate for immunotherapy A therapeutically attractive feature of non-mutant TAs is their sharing among patients. In contrast to patient-specific mTSA, melanoma-derived aeTSA was abundant and shared at the peptide and RNA levels (Figures 4A-4B). Nevertheless, TAs exhibited expression patterns and regulation specific to TA type, cancer type, and cancer subtype. Specifically, aeTSA encoded by carcinoembryonic (or oncogenic germ cell) genes and TAA encoded by cell cycle genes were found to be enriched in TCGA-derived melanoma samples, consistent with increased stem cell characteristics in these tumors (Figures 4B, 5).

[0143] Using TCGA multi-omics data, a significant correlation was found between TA expression and their corresponding source genes, suggesting regulation at the gene level (Figure 4C). aeTSA expression was frequently correlated with hypomethylation of source gene promoters in melanoma. In contrast, TAA and LSA showed universal associations with both local DNA copy number (CNV) and hypomethylation of source gene promoters, while TA type did not correlate with TMB (Figure 4D). In summary, these results, consistent with the role of MAP in mirroring internal cellular states, suggest that TA expression reflects a cancer cell program shared among patients and regulated at least partially at the mutational and / or epigenetic levels. The association with cancer (sub)type suggests that prioritization of non-mutant TAs may be possible and likely important for effective therapy.

[0144] MAPs obtained from MHC I immunoprecipitation of bulk tumor lysates are "contaminated" by tumor-infiltrating immune cells and peptides derived from other stromal cells in the microenvironment. 13Therefore, we aimed to verify that non-mutant TA expression is associated with malignant cells (or cell lineages of origin for LSA) using a publicly available single-cell RNA-seq dataset from melanoma. 14 (Figures 6A, 6B). TA was found to be highly and predominantly expressed by cancer cells (Figures 7A, 7B). Of the detected aeTSAs, most were expressed only in cancer cells (72%). When detected in annotated non-cancer cells, TA expression was associated with up to 100% cell doublet formation between non-cancer and cancer cells (Figure 8A). Indeed, melanoma TA-positive non-cancer cell populations showed increased expression of melanoma (and melanocyte) markers MLANA (Figure 8B, upper panel) and PMEL (Figure 8B, lower panel) compared with TA-negative non-cancer cells. Therefore, aeTSAs are cancer cell-specific, and their detection in other cell populations is due to technical limitations in single-cell sample preparation.

[0145] In summary, the results presented in this study strongly support the immunotargeting of non-mutant TAs across cancers with varying TMB levels. aeTSA is a particularly attractive target for immunotherapy given its cancer specificity, immunogenicity, high abundance, and patient-to-patient sharing.

[0146] While the present invention has been described herein by the specific embodiments described above, it may be modified without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word “including” is used as an open-ended term substantially equivalent to the phrase “including, but not limited to.” The singular forms “a,” “an,” and “the” include the corresponding plural subject matter unless the context clearly indicates otherwise.

[0147] References 1.Chong,C.et al.Integrated proteogenomic deep sequencing and analytics accurately identify non-canonical peptides in tumor immunopeptidomes.Nat.Commun.11,1293(2020). 2.Lamoliatte,F.,McManus,F.P.,Maarifi,G.,Chelbi-Alix,M.K.& Thibault,P.Uncovering the SUMOylation and ubiquitylation crosstalk in human cells using sequential peptide immunopurification.Nat.Commun.8,14109(2017). 3.Laumont,C.M.et al.Noncoding regions are the main source of targetable tumor-specific antigens.Sci.Transl.Med.10,eaau5516(2018). 4.Ehx,G.et al.Atypical acute myeloid leukemia-specific transcripts generate shared and immunogenic MHC class-I-associated epitopes Article Atypical acute myeloid leukemia-specific transcripts generate shared and immunogenic MHC class-I-associated epitopes.Immunity 54,737-752(2021). 5.Reynisson,B.,Alvarez,B.,Paul,S.,Peters,B.& Nielsen,M.NetMHCpan-4.1 and NetMHCIIpan-4.0:improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data.Nucleic Acids Res.48,W449-W454(2020). 6.Courcelles,M.et al.MAPDP:A Cloud-Based Computational Platform for Immunopeptidomics Analyses.J.Proteome Res.19,1873-1881(2020). 7.Ruiz Cuevas,M.V.et al.BamQuery:a proteogenomic tool for the genome-wide exploration of the immunopeptidome.bioRxiv(2022).doi:10.1101 / 2022.10.07.510944. 8.Riaz,N.et al.Tumor and Microenvironment Evolution during Immunotherapy with Nivolumab.Cell 171,934-949.e15(2017). 9.Danilova,L.et al.The mutation-associated neoantigen functional expansion of specific T cells(MANAFEST) assay:A sensitive platform for monitoring antitumor immunity.Cancer Immunol.Res.6,888-899 (2018). 10.Gurjao,C.,Tsukrov,D.,Imakaev,M.,Luquette,L.J.& Mirny,L.A.Is tumor mutational burden predictive of response to immunotherapy? Elife(2023).doi:10.7554 / eLife.87465.1. 11.Gide,T.N.et al.Distinct Immune Cell Populations Define Response to Anti-PD-1 Monotherapy and Anti-PD-1 / Anti-CTLA-4 Combined Therapy.Cancer Cell 35,238-255.e6(2019). 12.Du,K.et al.Pathway signatures derived from on-treatment tumor specimens predict response to anti-PD1 blockade in metastatic melanoma.Nat.Commun.12,6023(2021). 13.Jaeger,A.M.et al.Deciphering the immunopeptidome in vivo reveals new tumour antigens.Nature 607,149-155(2022). 14.Zhang,C.et al.A single-cell analysis reveals tumor heterogeneity and immune environment of acral melanoma.Nat.Commun.13,7250(2022). 15.Apavaloaei,A.et al.Induced pluripotent stem cells display a distinct set of MHC I-associated peptides shared by human cancers.Cell Rep.40,111241(2022). 16.Zhao,Q.et al.Proteogenomics Uncovers a Vast Repertoire of Shared Tumor-Specific Antigens in Ovarian Cancer.Cancer Immunol.Res.8,544-555(2020). 17.Huang,X.et al.Insulin-like growth factor 2 mRNA-binding protein 1(IGF2BP1)in cancer.J.Hematol.Oncol.11,88(2018). 18.Miranda,A.et al.Cancer stemness,intratumoral heterogeneity,and immune response across cancers.Proc.Natl.Acad.Sci.U.S.A.116,9020-9029(2019). 19.Van Allen,E.M.et al.Genomic correlates of response to CTLA-4 blockade in metastatic melanoma.Science 352,207-212(2015). 20.Hugo,W.et al.Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma.Cell 165,35-44(2016). 21.Liu,D.et al.Integrative molecular and clinical modeling of clinical outcomes to PD1 blockade in patients with metastatic melanoma.Nat.Med.25,1916-1927(2019).

Claims

1. Tumor antigen peptides (TAPs) that contain or consist of one of the amino acid sequences defined in SEQ ID NOs: 1 to 505.

2. The TAP according to claim 1, wherein the TAP is bound to an HLA-A*01:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 11, 14, 20, 27, 29, 37, 86, 87, 121, 150, 165, 173, 279, 288, 299, 308, 312, 316, 319, 331, 340, 352, 362, 363, 384, 395, 398, 411, 419, 429, 458, 474, 476, 487, 492, 500, or 501.

3. The aforementioned TAP binds to the HLA-A*02:01 molecule, and SEQ ID NOs: 1, 2, 4, 19, 22, 25, 40, 50, 52, 54, 60, 65, 67, 76, 81, 89, 91, 103, 107, 120, 124, 128, 135, 157, 175, 179, 186, 188, 201, 208, 218, 219, 228, 232, 233, 240, 250, 2 The TAP according to claim 1, comprising or consisting of the sequence 57, 261, 263, 265, 266, 282, 286, 298, 311, 315, 317, 325, 333, 337, 349, 367, 373, 387, 388, 393, 399, 404, 412, 414, 415, 422, 434, 437, 455, 480, 489, or 494.

4. The TAP according to claim 1, wherein the TAP is bound to an HLA-A*02:09 molecule and comprises or consists of the sequence of SEQ ID NOs: 22, 40, 50, 62, 83, 89, 232, 265, 282, 311, 315, 317, 337, 349, 367, 399, 407, 412, or 494.

5. The TAP according to claim 1, wherein the TAP is bound to an HLA-A*03:01 molecule and includes or consists of the sequence of SEQ ID NOs: 209, 247, 272, 347, or 505.

6. The TAP according to claim 1, wherein the TAP is bound to an HLA-A*23:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 48, 84, 129, 151, 152, 171, 174, 182, 223, 237, 245, 290, 358, 397, 426, 446, 481, 482, or 486.

7. The TAP according to claim 1, wherein the TAP is bound to an HLA-A*24:02 molecule and includes or consists of the sequence of SEQ ID NO: 31, 244, or 481.

8. The TAP according to claim 1, wherein the TAP is bound to an HLA-A*25:01 molecule and includes or consists of the sequence of SEQ ID NOs: 11, 31, 249, 289, 403, 425, 464, or 490.

9. The TAP according to claim 1, wherein the TAP is bound to an HLA-A*26:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 11, 20, 27, 39, 41, 42, 45, 114, 116, 122, 168, 181, 268, 284, 329, 364, 398, 417, 430, 440, 447, 452, 457, 460, 462, 475, 490, or 497.

10. The TAP according to claim 1, wherein the TAP is bound to an HLA-A*30:01 molecule and includes or consists of the sequence of SEQ ID NO: 26, 304, or 376.

11. The TAP according to claim 1, wherein the TAP is bound to an HLA-A*30:02 molecule and includes or consists of the sequence of SEQ ID NOs: 117, 185, 190, 193, 197, 256, 294, 323, 471, or 485.

12. The TAP according to claim 1, wherein the TAP is bound to an HLA-A*33:01 molecule and contains or consists of the sequence of Sequence ID No.

248.

13. The aforementioned TAP binds to the HLA-A*68:01 molecule, and SEQ ID NOs: 6-8, 12, 15, 23, 28, 41, 42, 47, 51, 53, 56, 59, 64, 66, 68, 70, 73-75, 77, 79, 80, 82, 85, 88, 92, 93, 96-99, 101, 102, 108-112, 118, 1 19, 123, 130, 131, 134, 137, 138, 141, 142, 144-147, 149, 153, 154, 156, 158, 160, 161, 166, 169, 172, 176, 184, 198, 202, 203, 212, 214, 216, 217, 220, 224, 226, 229, 2 34, 236, 239, 248, 254, 269, 270, 275, 280, 283, 287, 293, 300, 301, 303, 306, 310, 314, 320, 328, 332, 348, 354, 355, 359, 360, 366, 369, 370, 372, 381, 382, ​​386, 389, The TAP according to claim 1, comprising or consisting of the sequence 390, 392, 394, 402, 405, 406, 409, 413, 418, 424, 428, 436, 441-445, 449, 456, 459, 465-468, 473, 479, 483, 488, 493, 499, or 502.

14. The TAP according to claim 1, wherein the TAP is bound to an HLA-B*07:02 molecule and includes or consists of the sequence of SEQ ID NOs: 35, 36, 43, 104, 125, 127, 139, 140, 187, 191, 196, 206, 231, 243, 246, 251, 252, 260, 281, 291, 292, 321, 330, 361, 408, 432, 435, 439, 451, 461, 491, or 503.

15. The TAP according to claim 1, wherein the TAP is bound to an HLA-B*08:01 molecule and includes or consists of the sequence of SEQ ID NOs: 21, 162, 189, 286, 307, 309, 317, 339, 343, 349, or 414.

16. The TAP according to claim 1, wherein the TAP is bound to an HLA-B*13:02 molecule and includes or consists of the sequence of SEQ ID NOs. 207, 311, 393, 400, or 455.

17. The TAP according to claim 1, wherein the TAP is bound to an HLA-B*14:01 molecule and contains or consists of the sequence of Sequence ID No.

396.

18. The TAP according to claim 1, wherein the TAP is bound to an HLA-B*15:01 molecule and includes or consists of the sequence of SEQ ID NOs: 11, 26, 30, 63, 113, 132, 148, 192, 195, 200, 207, 210, 222, 235, 238, 258, 262, 267, 276, 295, 297, 305, 309, 318, 326, 336, 342, 374, 377, 383, 401, 421, 427, 433, 438, 450, 463, 470, or 477.

19. The TAP according to claim 1, wherein the TAP is bound to an HLA-B*18:01 molecule and includes or consists of the sequence of SEQ ID NOs: 3, 10, 33, 225, or 396.

20. The TAP according to claim 1, wherein the TAP is bound to an HLA-B*37:01 molecule and includes or consists of the sequence of SEQ ID NO:

302.

21. The TAP according to claim 1, wherein the TAP is bound to an HLA-B*38:01 molecule and includes or consists of the sequence of SEQ ID NOs: 26, 180, 338, 341, 378, 380, 416, 448, 454, 496, or 498.

22. The TAP according to claim 1, wherein the TAP is bound to an HLA-B*40:01 molecule and includes or consists of the sequence of SEQ ID NO: 215 or 322.

23. The TAP according to claim 1, wherein the TAP is bound to an HLA-B*40:02 molecule and includes or consists of the sequence of SEQ ID NOs: 213, 215, 339, or 351.

24. The TAP according to claim 1, wherein the TAP is bound to an HLA-B*44:02 molecule and includes or consists of the sequence of SEQ ID NOs: 346, 353, 356, or 453.

25. The TAP according to claim 1, wherein the TAP is bound to an HLA-B*49:01 molecule and includes or consists of the sequence of SEQ ID NOs: 9, 57, 78, 115, 136, 167, 255, 273, 274, 278, 313, 339, 480, 484, or 495.

26. The TAP according to claim 1, wherein the TAP is bound to an HLA-B*51:01 molecule and includes or consists of the sequence of SEQ ID NO: 227 or 259.

27. The TAP according to claim 1, wherein the TAP is bound to an HLA-C*03:03 molecule and comprises or consists of the sequence of SEQ ID NOs: 24, 69, 204, or 388.

28. The TAP according to claim 1, wherein the TAP is bound to an HLA-C*03:04 molecule and comprises or consists of the sequence of SEQ ID NOs: 24, 49, 69, 159, 241, 242, or 334.

29. The TAP according to claim 1, wherein the TAP is bound to an HLA-C*06:02 molecule and includes or consists of the sequence of SEQ ID NOs: 13, 34, or 72.

30. The TAP according to claim 1, wherein the TAP is bound to an HLA-C*07:01 molecule and includes or consists of the sequence of SEQ ID NOs: 13, 75, 133, 163, 183, 199, 205, 285, 334, 391, or 472.

31. The TAP according to claim 1, wherein the TAP is bound to an HLA-C*07:02 molecule and comprises or consists of the sequence of SEQ ID NOs: 13, 32, 36, 105, 177, 339, 371, or 478.

32. The TAP according to claim 1, wherein the TAP is bound to an HLA-C*08:02 molecule and comprises or consists of the sequence of SEQ ID NO: 18, 54, or 379.

33. The TAP according to claim 1, wherein the TAP is bound to an HLA-C*12:03 molecule and comprises or consists of the sequence of SEQ ID NOs: 24, 49, 58, 71, 94, 162, 178, 242, 289, 344, 345, 357, 367, 368, 375, 423, or 431.

34. The TAP according to claim 1, wherein the TAP is bound to an HLA-C*14:02 molecule and includes or consists of the sequence of SEQ ID NOs: 105, 271, 350, 365, 385, 410, 423, or 504.

35. The TAP according to any one of claims 1 to 34, which is encoded by a sequence located in a non-protein-coding region of the genome or by a long non-coding RNA.

36. The TAP according to claim 35, wherein the non-protein coding region of the genome is an intergeneric region.

37. The TAP according to claim 35, wherein the non-protein coding region of the genome is an intron.

38. The TAP according to any one of claims 1 to 37, wherein the TAP is conjugated with a molecule that increases the protease resistance, plasma protein binding, plasma half-life, and / or intracellular osmosis of the TAP.

39. A combination comprising at least two of the TAPs or nucleic acids defined in any one of claims 1 to 38.

40. A synthetic long peptide (SLP) comprising at least one of the amino acid sequences defined in claim 1.

41. A nucleic acid encoding one or more of the TAPs described in claims 1 to 38, the combinations described in claim 39, or the SLPs described in claim 40.

42. The nucleic acid according to claim 41, wherein the nucleic acid is mRNA, and the mRNA optionally comprises one or more 5'-terminal modifications, 3'-terminal modifications, and / or modified nucleosides to increase the stability of the mRNA, improve translation, and / or reduce immunogenicity.

43. The nucleic acid according to claim 41, wherein the nucleic acid is DNA.

44. The nucleic acid according to any one of claims 41 to 43, wherein the nucleic acid is a component of a viral vector.

45. A vesicle or particle comprising a TAP according to any one of claims 1 to 38, a combination according to claim 39, an SLP according to claim 40, or a nucleic acid according to any one of claims 41 to 44.

46. The vesicle or particle according to claim 45, wherein the vesicle is a lipid nanoparticle (LNP).

47. A vesicle or particle according to claim 45 or 46, comprising a cationic lipid.

48. A composition comprising a TAP according to any one of claims 1 to 38, a combination according to claim 39, an SLP according to claim 40, a nucleic acid according to any one of claims 41 to 44, or a vesicle or particle according to any one of claims 45 to 47, and a pharmaceutically acceptable carrier.

49. A vaccine comprising a TAP according to any one of claims 1 to 38, a combination according to claim 39, an SLP according to claim 40, a nucleic acid according to any one of claims 41 to 44, a vesicle or particle according to any one of claims 45 to 47, or a composition according to claim 48, and an adjuvant.

50. An isolated major histocompatibility complex (MHC) class I molecule comprising a TAP according to any one of claims 1 to 38 within its peptide bond groove.

51. An isolated MHC class I molecule according to claim 50, which is in the form of a polymer.

52. The isolated MHC class I molecule according to claim 51, wherein the polymer is a tetramer.

53. Isolated cells comprising (i) a TAP according to any one of claims 1 to 38, (ii) a combination according to claim 39, (iii) an SLP according to claim 40, (iv) a nucleic acid according to any one of claims 41 to 44, or (v) a vector comprising a nucleotide sequence encoding a TAP according to any one of claims 1 to 38, a combination according to claim 39, or an SLP according to claim 40.

54. Isolated cells expressing, on their surface, a major histocompatibility complex (MHC) class I molecule comprising a TAP or combination described in any one of claims 1 to 38 within a peptide bond groove.

55. The cell according to claim 53 or 54, which is an antigen-presenting cell (APC).

56. The cell according to claim 55, wherein the APC is a dendritic cell.

57. A T cell receptor (TCR) or nucleic acid encoding the TCR, which specifically recognizes an isolated MHC class I molecule according to any one of claims 50 to 52, and / or an MHC class I molecule expressed on the surface of a cell according to any one of claims 54 to 56.

58. The TCR according to claim 57, which is a soluble TCR.

59. An antibody or an antigen-binding fragment thereof, or a nucleic acid encoding the antibody or antigen-binding fragment, which specifically binds to an isolated MHC class I molecule according to any one of claims 50 to 52, and / or an MHC class I molecule expressed on the surface of a cell according to any one of claims 54 to 56.

60. A bispecific TCR, or a bispecific antibody, or an antigen-binding fragment thereof, as described in claim 57 or 58, or an antibody or antigen-binding fragment thereof as described in claim 59.

61. The TCR, antibody or antigen-binding fragment according to claim 60, wherein the bispecific antibody or its antigen-binding fragment is a single-stranded diabody (scDb).

62. The TCR, antibody, or antigen-binding fragment according to claim 60 or 61, wherein the bispecific TCR, antibody, or antigen-binding fragment also specifically binds to T cell signaling molecules.

63. The TCR, antibody or antigen-binding fragment according to claim 62, wherein the T cell signaling molecule is a CD3 chain.

64. A chimeric antigen receptor (CAR), or a nucleic acid encoding the CAR, comprising the antibody or antigen-binding fragment thereof as described in claim 59.

65. Isolated cells expressing the TCR described in claim 57 or the CAR described in claim 64 on their cell surface.

66. CD8 ＋ The isolated cells according to claim 65, which are T lymphocytes.

67. A cell population comprising at least 0.5% isolated cells as defined in claim 65 or 66.

68. A method for treating cancer in a subject, wherein an effective amount of the subject (a) A TAP containing or consisting of any one of the sequences defined in SEQ ID NOs: 1 to 505, or any combination thereof, or a synthetic long peptide (SLP) containing at least one of the sequences defined in SEQ ID NOs: 1 to 505. (b) A TAP as defined in (a), a combination thereof, or at least one nucleic acid encoding an SLP, (c) A vesicle or particle containing a TAP as defined in (a), a combination thereof, or an SLP, or at least one nucleic acid as defined in (b), (d) A composition comprising a TAP as defined in (a), a combination thereof, or an SLP, at least one nucleic acid as defined in (b), or a vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier. (e) A vaccine comprising a TAP as defined in (a), a combination thereof, or an SLP, at least one nucleic acid as defined in (b), a vesicle or particle as defined in (c), or a composition as defined in (d), and an adjuvant. (f) Cells expressing major histocompatibility complex (MHC) class I molecules on their surface that contain TAPs as defined in (a) or a combination thereof within their peptide bond grooves, A cell expressing a T cell receptor (TCR) or chimeric antigen receptor (CAR) on its cell surface that specifically recognizes MHC class I molecules expressed on the surface of the cell as defined in (g)(f), or (h)(f) A soluble TCR, antibody or its antigen-binding fragment, or CAR, or a nucleic acid encoding the soluble TCR, antibody, its antigen-binding fragment, or CAR, which specifically binds to the MHC class I molecule expressed on the surface of the cell defined in (h)(f). A method including administering [a substance].

69. The method according to claim 68, wherein the cancer is melanoma.

70. The method according to claim 68 or 69, further comprising administering at least one additional antitumor agent or therapy to the subject.

71. The method according to claim 70, wherein the at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiotherapy, or surgery.

72. For the manufacture of pharmaceuticals for treating cancer in a subject, or for the manufacture of pharmaceuticals for treating cancer in a subject, (a) A TAP containing or consisting of any one of the sequences defined in SEQ ID NOs: 1 to 505, or any combination thereof, or a synthetic long peptide (SLP) containing at least one of the sequences defined in SEQ ID NOs: 1 to 505. (b) A TAP as defined in (a), a combination thereof, or at least one nucleic acid encoding an SLP, (c) A vesicle or particle containing a TAP as defined in (a), a combination thereof, or an SLP, or at least one nucleic acid as defined in (b), (d) A composition comprising a TAP as defined in (a), a combination thereof, or an SLP, at least one nucleic acid as defined in (b), or a vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier. (e) A vaccine comprising a TAP as defined in (a), a combination thereof, or an SLP, at least one nucleic acid as defined in (b), a vesicle or particle as defined in (c), or a composition as defined in (d), and an adjuvant. (f) Cells expressing major histocompatibility complex (MHC) class I molecules on their surface that contain TAPs as defined in (a) or a combination thereof within their peptide bond grooves, A cell expressing a T cell receptor (TCR) or chimeric antigen receptor (CAR) on its cell surface that specifically recognizes MHC class I molecules expressed on the surface of the cell as defined in (g)(f), or (h)(f) A soluble TCR, antibody or its antigen-binding fragment, or CAR, or a nucleic acid encoding the soluble TCR, antibody, its antigen-binding fragment, or CAR, which specifically binds to the MHC class I molecule expressed on the surface of the cell defined in (h)(f). Use.

73. The use according to claim 72, wherein the cancer is melanoma.

74. The use according to claim 72 or 73, further comprising using at least one additional antitumor agent or therapy on the subject.

75. The use according to claim 74, wherein the at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiotherapy, or surgery.

76. A drug used to treat cancer in the subject, wherein the drug is (a) A TAP containing or consisting of any one of the sequences defined in SEQ ID NOs: 1 to 505, or any combination thereof, or a synthetic long peptide (SLP) containing at least one of the sequences defined in SEQ ID NOs: 1 to 505. (b) A TAP as defined in (a), a combination thereof, or at least one nucleic acid encoding an SLP, (c) A vesicle or particle containing a TAP as defined in (a), a combination thereof, or an SLP, or at least one nucleic acid as defined in (b), (d) A composition comprising a TAP as defined in (a), a combination thereof, or an SLP, at least one nucleic acid as defined in (b), or a vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier. (e) A vaccine comprising a TAP as defined in (a), a combination thereof, or an SLP, at least one nucleic acid as defined in (b), a vesicle or particle as defined in (c), or a composition as defined in (d), and an adjuvant. (f) Cells expressing major histocompatibility complex (MHC) class I molecules on their surface that contain TAPs as defined in (a) or a combination thereof within their peptide bond grooves, A cell expressing a T cell receptor (TCR) or chimeric antigen receptor (CAR) on its cell surface that specifically recognizes MHC class I molecules expressed on the surface of the cell as defined in (g)(f), or (h)(f) A soluble TCR, antibody or its antigen-binding fragment, or CAR, or a nucleic acid encoding the soluble TCR, antibody, its antigen-binding fragment, or CAR, which specifically binds to the MHC class I molecule expressed on the surface of the cell defined in (h)(f). It is a drug.

77. The agent for use according to claim 76, wherein the cancer is melanoma.

78. The agent for use according to claim 76 or 77, further comprising using at least one additional antitumor agent or therapy on the subject.

79. The agent for use according to claim 78, wherein the at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiotherapy, or surgery.

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